Methods and compositions for modulating t cell and/or b cell activation

ABSTRACT

The present invention provides methods of reducing or enhancing T cell activation and/or B cell activation in a subject, comprising administering to a subject an effective amount of an inhibitor or enhancer, respectively, of Semaphorin 6D (Sema6D) activity on T cells and/or B cells.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S.Provisional Application No. 60/785,310, filed Mar. 23, 2006, the entirecontents of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

Aspects of this invention were supported by government finds provided bythe National Institutes of Health Grant No. RO1-AI-29564. The U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to regulating immune responses bymodulating T cell activation and/or B cell activation.

BACKGROUND ART

The immune system is comprised of a complex network of autonomous cellsworking together to manage a chaotic situation. The regulation of theimmune network is equally complex, characterized by hubs of activationcontrol. The dendritic cell (DC) represents one hub of immune controlvia its unique abilities to regulate activation of CD4⁺ T cells. Inturn, CD4⁺ T cells are able to affect the activity of a wide variety ofimmune cells of both the adaptive and innate categories. During aninitial interaction, DCs present antigen (Ag) in the context of MHCclass II molecules for recognition by CD4⁺ T cells via their T cellreceptor (TCR). Binding of Ag by the TCR represents signal one, butfurther stimulation derived from costimulatory signals is required forfill activation of T cells via DCs. Many co-stimulatory receptor-ligandpairs have been identified between T cells and DCs. The prototypicalcostimulation interacting pairs on T cells and DCs are CD154-CD40 andCD28-B7 (T-DC respectively). Since their identification, experimentalmanipulation of these receptors and ligands has proven to be a powerfultool in modulating a wide variety of immune responses ranging fromtransplant tolerance and autoimmunity to tumor rejection (1-7).

The present invention overcomes previous shortcomings in the art bydemonstrating that PlexA1 expressed on DCs and Sema6D expressed on Tcells (e.g., CD4⁺ T cells) and on B cells, represent a novelreceptor-ligand costimulation pair, capable of regulating immune systemactivity.

SUMMARY OF THE INVENTION

The present invention provides a method of reducing T cell activation ina subject, comprising administering to the subject an effective amountof an inhibitor of Semaphorin 6D (Sema6D) activity on T cells.

Further provided herein is a method of increasing T cell activation in asubject, comprising administering to the subject an effective amount ofan enhancer of Semaphorin 6D (Sema6D) activity on T cells.

In addition, the present invention provides a method of identifying anactivated T cell, comprising detecting Sema6D on the surface of the Tcell.

The present invention also provides a method of monitoring T cellactivation over time, comprising detecting Sema6D on the surface of a Tcell over time and measuring changes in the amount of Sema6D on thesurface of a T cell over time.

In further embodiments, the present invention provides a method ofidentifying a substance having the ability to inhibit Sema6D activity,comprising contacting the substance with T cells under conditionswherein Sema6D activity can occur and measuring the amount of Sema6Dactivity in the presence and in the absence of the substance; whereby adecrease in Sema6D activity in the presence of the substance as comparedto the amount of Sema6D activity in the absence of the substanceidentifies a substance having the ability to inhibit Sema6D activity.

Additionally provided is a method of identifying a substance having theability to enhance Sema6D activity, comprising contacting the substancewith T cells under conditions whereby Sema6D activity can occur andmeasuring the amount of Sema6D activity in the presence and in theabsence of the substance; whereby an increase in Sema6D activity in thepresence of the substance as compared to the amount of Sema6D activityin the absence of the substance identifies a substance having theability to enhance Sema6D activity.

Further provided herein is a method of reducing B cell activation in asubject, comprising administering to a subject in need of reduced B cellactivation an effective amount of an inhibitor of Semaphorin 6D (Sema6D)activity on B cells.

In further embodiments, the present invention provides a method ofincreasing B cell activation in a subject, comprising administering to asubject in need of increased B cell activation an effective amount of anenhancer of Semaphorin 6D (Sema6D) activity on B cells.

Also provided herein is a method of identifying an activated B cell,comprising detecting Sema6D on the surface of the B cell and a method ofidentifying an activated B cell, comprising detecting messenger RNAencoding Sema6D in the B cell.

Additional embodiments include a method of monitoring B cell activationover time, comprising detecting Sema6D on the surface of a B cell overtime and measuring changes in the amount of Sema6D on the surface of a Bcell over time, as well as a method of monitoring B cell activation overtime, comprising detecting messenger RNA encoding Sema6D in a B cellover time and measuring changes in the amount of messenger RNA encodingSema6D in the B cell over time.

Additionally provided herein is a method of identifying a substancehaving an inhibitory effect on Sema6D activity and/or having aninhibitory effect on B cell activation, comprising contacting thesubstance with B cells under conditions whereby Sema6D activity and/or Bcell activation can occur and measuring the amount of Sema6D activityand/or B cell activation in the presence and in the absence of thesubstance; whereby a decrease in Sema6D activity and/or B cellactivation in the presence of the substance as compared to the amount ofSema6D activity and or/B cell activation in the absence of the substanceidentifies a substance having the ability to inhibit Sema6D activityand/or B cell activation.

Furthermore, the present invention provides a method of identifying asubstance having an enhancing effect on Sema6D activity and/or B cellactivation, comprising contacting the substance with B cells underconditions whereby Sema6D activity and/or B cell activation can occurand measuring the amount of Sema6D activity and/or B cell activation inthe presence and in the absence of the substance; whereby an increase inSema6D activity and/or B cell activation in the presence of thesubstance as compared to the amount of Sema6D activity and/or B cellactivation in the absence of the substance identifies a substance havingthe ability to enhance Sema6D activity and/or B cell activation.

It is further contemplated herein that the present invention provides amethod of treating a B cell-related disorder and/or a T cell relateddisorder and/or other white blood cell-related disorder in a subject,comprising administering to the subject a therapeutic amount of aninhibitor of Semaphorin 6D (Sema6D) activity on B cells, T cells and/orother white blood cells.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of Sema6D mRNA in the immune system. (a)SymAtlas gene array mouse cell and tissue expression of Sema6D mRNA. (b)SymAtlas gene array human cell and tissue expression of Sema6D mRNA. (c)SymAtlas gene array human cancer cell expression of Sema6D mRNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to thefollowing embodiments. As is apparent by these descriptions, thisinvention can be embodied in different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Except as otherwise indicated, standard methods can be used for theproduction of viral and non-viral vectors, manipulation of nucleic acidsequences, production of transformed cells, and the like according tothe present invention. Such techniques are known to those skilled in theart. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al. CURRENTPROTOCOLS 1N MOLECULAR BIOLOGY (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

The present invention is based on the unexpected discovery that thesemaphorin 6D protein on a T cell (e.g., CD4⁺ T cell) surface is aligand for the Plexin-A1 (PlexA1) receptor protein on antigen-presentingcells, thereby providing the first identification of a ligand for aPlexin-A1 receptor on an immune system cell. Thus, in one embodiment,the present invention provides a method of reducing or inhibiting T cellactivation in a subject, comprising administering to the subject (e.g.,a subject in need of reduced T cell activation) an effective amount ofan inhibitor of Semaphorin 6D (Sema6D) activity on CD4⁺ T cells.

The present invention additionally provides a method of reducing orinhibiting B cell activation in a subject, comprising administering to asubject in need of reduced B cell activation an effective amount of aninhibitor of Semaphorin 6D (Sema6D) activity on B cells.

In the methods of this invention whereby T cell activation and/or B cellactivation is reduced or inhibited, a subject of these methods caninclude a subject having, or at risk of having, an autoimmune disorderor disease, a transplant recipient, a subject having an inflammatoryresponse or at risk of having an inflammatory response, a subject havingan allergic response or at risk of having an allergic response and anysubject in whom it is desirable to suppress an immune responseassociated with T cell activation and/or B cell activation, as known inthe art.

According to the methods of this invention, an inhibitor of Sema6Dactivity can be, but is not limited to an antibody or antibody fragmentthat specifically binds Sema6D, a fusion protein comprising theextracellular domain of the Sema6D protein and an immunoglobulinfragment, an antibody or antibody fragment that specifically bindsPlexA1, small molecule mimetics that block the binding of Sema6D toPlexA1 and any substance that inhibits binding of Sema6D to PlexA1 asnow known or later identified.

Also included in the methods described herein is a substance thatreduces or inhibits Sema6D activity and/or PlexA1 activity at thetranscriptional, post-transcriptional, translational and/orpost-translational level. For example, the transcription factor class IItransactivator (CIITA) can activate the PlexA1 gene expression in immunedendritic cells (Nature Immunol. 4(9):891-8 2003) and this method ofactivation is not likely to occur in other tissues with PlexA1 such asneurons and heart cells, since CIITA is not expressed in these othercells. It would be possible to alter CIITA to induce a change in PlexA1expression predominantly in immune dendritic cells.

In further embodiments of this invention, the inhibitor of Sema6Dactivity and/or inhibitor of PlexA1 activity is administered incombination (either before, after and/or simultaneously) with anotheranti-T cell therapeutic and/or anti-B cell therapeutic, eithersimultaneously, before and/or after administration of the inhibitor ofSema6D activity and/or inhibitor of PlexA1 activity. Nonlimitingexamples of an anti-T cell therapeutic include an antibody or fragmentthereof or other ligand or fragment thereof that specifically bindsand/or inhibits activity of CD3 protein, CD40 protein, B7 familyproteins, and/or CD28 family proteins; cyclosporine; FK504; steroids;and/or substances that target MHC-1 and/or MHC-II molecules,immunosuppressive drugs, interferons, corticosteroids, azathioprine,cyclophosphamide, etc. Also included are anti-T cell therapeutics thatreduce or inhibit CD3 (e.g., OKT®3 monoclonal antibody), CD40, B7 and/orCD28 activity in T cells at the transcriptional, post-transcriptional,translational and/or post-translational level (e.g., an antisensenucleic acid that binds a coding sequence of the Sema6D protein, aninterfering RNA that inhibits or suppresses transcription and/ortranslation of the Sema6D protein, a ribozyme, etc.), therapies thattarget T cell activation transcription factors, such as inhibitors ofIκB kinase (IKK), which would also inhibit the transcription factor,Nuclear Factor kappa light chain enhancer in B cells (NF-κb), orcyclosporine, which inhibits the calcineurin pathway important for theactivation of the transcription factor, Nuclear Factor of Activated Tcells). Also included are Basiliximab (anti-CD25), Alefacept (LFA3-Igfusion; blocks CD2), Daclizumab (Anti-CD25), Tysabri (anti-VLA4) andanti-CLA4 Ab. Other inhibitors that can be used in the methods of thisinvention include but are not limited to Omalizumab (Anti-IgE mab;targets mast cells and basophils) and Lumiliximab (anti-CD23; targetsmast cells and basophils).

Nonlimiting examples of an anti-B cell therapeutic include an antibodyor fragment thereof or other ligand or fragment thereof thatspecifically binds and/or inhibits activity of CD20 protein (e.g.,Rituximab® monoclonal antibody), immunosuppressive drugs, interferons,corticosteroids, azathioprine, cyclophosphamide, CTLA4-IG (targetsCD80/86 on DCs and B cells), Belimumab (targets Blys (BAFF) interactionswith receptors on B cells), and Natalizumab or Tysabri (Anti-VLA4;targets T cells and B cells),

Further provided is a method of increasing T cell and/or B cellactivation in a subject, comprising administering to the subject (e.g.,a subject in need of increased T cell activation and/or increased B cellactivation) an effective amount of an enhancer of Semaphorin 6D (Sema6D)activity on T cells and/or B cells.

In the methods provided herein for enhancing T cell activation and/or Bcell activation, a subject can be a subject having an infection or atrisk of having an infection, a subject having a suppressed immune systemor suppressed immune response or at risk of having a suppressed immunesystem or suppressed immune response, as known in the art. Examples ofinfections that cause immunosuppression include but are not limited tohuman immunodeficiency virus infection, cytomegalovirus infection,vaccinia virus infection, and F. tularenesis bacterial infection.Conditions under which immune suppression occurs include severeimmunodeficiencies, advanced age, chemotherapy, radiation therapy,irradiation and upon severe burn. In additional embodiments, theenhancer of T cell activation and/or B cell activation can beadministered in combination (either before, after and/or simultaneously)with a T cell activation therapeutic and/or a B cell activationtherapeutic. Nonlimiting examples of a T cell activation and/or a B cellactivation therapeutic of this invention include vaccines such aspeptides, DNA and glycoproteins and adjuvants such as toll-like receptoragonists, and the Bacillus Calmette-Guerin.

It is further contemplated herein that T cell activation and/or B cellactivation can be reduced, inhibited or enhanced in methods employing exvivo T cells and/or B cells and/or antigen presenting cells that havebeen removed from a subject and are subsequently administered to thesame subject or a different subject of the same species. Thus, thepresent invention provides a method of enhancing T cell activationand/or B cell activation, comprising contacting a T cell and/or a B cellwith an enhancer of Sema6D activity and/or an enhancer of PlexA1activity in the presence of an antigen presenting cell having PlexA1 onthe surface, under conditions whereby T cell activation and/or B cellactivation can occur and then administering the activated T cell and/oractivated B cell and/or antigen presenting cell to a subject. Furtherprovided is a method of reducing T cell activation and/or B cellactivation, comprising contacting a T cell and/or B cell with aninhibitor of Sema6D activity and/or an inhibitor of PlexA1 activity inthe presence of an antigen presenting cell having PlexA1 on the surface,under conditions whereby inhibition of T cell activation and/orinhibition of B cell activation can occur and then administering the Tcell and/or B cell and/or antigen presenting cell to a subject.

In other embodiments, the present invention provides a method ofidentifying an activated T cell or activated B cell, comprisingdetecting Sema6D on the surface of the T cell or B cell. Furtherprovided herein is a method of identifying an activated T cell oractivated B cell, comprising detecting messenger RNA encoding Sema6D inthe T cell or B cell.

In methods of this invention wherein Sema6D is detected on the surfaceof a T cell or a B cell, such detection can be carried out according tomethods standard in the art for detecting a protein on the surface, of acell and such methods can be qualitative and/or quantitative.Furthermore, in methods of this invention wherein an amount of messengerRNA encoding Sema6D is detected, such detection can be carried outaccording to standard methods for detecting nucleic acid in a cell(e.g., polymerase chain reaction (PCR) and other nucleic acidamplification protocols, real-time PCR, RNase protection, in situhybridization, Northern blots, etc.) and such methods can be qualitativeand/or quantitative.

Thus, in some embodiments, the identification of an activated T cell oractivated B cell can be carried out by identifying an increase in theamount of Sema6D on the surface of a cell relative to a cell that is notactivated. An amount of Sema6D on a T cell or B cell that is notactivated can be determined by identifying T cells or B cells that arenot activated (as determined by features other than the absence ofSema6D, such as the absence of CD69, CD25, HLA-DR, CD62L, CD154 and/orCD44CD25, IL-2 production, ZAP70, LAT and Lck phosphorylation in Tcells) and measuring the amount of Sema6D on the surface of saidnon-activated cells to establish a baseline amount of Sema6D. Thus, anactivated T cell or activated B cell would be identified as having anamount of Sema6D on the surface that is increased relative to thebaseline amount.

Thus, in some embodiments, the increase in Sema6D protein can be anincrease of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%, 10%, 12%,15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190%, 200%, 300%, etc., relative to the amount of Sema6D proteinon the surface of a nonactivated T cell or nonactivated B cell.

In addition, the increase in Sema6D protein can be at least about 0.1fold, 0.2 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 2.5 fold, 3.0fold, 3.5 fold, 4.0 fold, 4.5 fold, 5.0 fold, 5.5 fold, 6.0 fold, 6.5fold, 7.0 fold, 7.5 fold, 8.0 fold, 8.5 fold, 9.0 fold, 9.5 fold, 10fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, 100 fold, etc., relative to the amount of Sema6D protein on thesurface of a nonactivated T cell or nonactivated B cell.

In other embodiments, the increase in Sema6D activity can be an increaseof at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%, 10%, 12%, 15%,18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 300%; etc., relative to the amount of Sema6D protein on thesurface of a nonactivated T cell or non activated B cell.

In addition, the increase in Sema6D activity can be at least about 0.1fold, 0.2 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 2.5 fold, 3.0fold, 3.5 fold, 4.0 fold, 4.5 fold, 5.0 fold, 5.5 fold, 6.0 fold, 6.5fold, 7.0 fold, 7.5 fold, 8.0 fold, 8.5 fold, 9.0 fold, 9.5 fold, 10fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, 100 fold, etc., relative to the amount of Sema6D protein on thesurface of a nonactivated T cell or nonactivated B cell.

Furthermore, in methods wherein the amount of mRNA encoding Sema6D ismeasured to identify an activated T cell or activated B cell, a baselineamount of mRNA in a nonactivated T cell or nonactivated B cell can bedetermined and an activated T cell or activated B cell can be identifiedby measuring the amount of mRNA relative to the baseline amount.

Thus, the increase in Sema6D mRNA can be of at least about 1%, 2%, 3%,4%, 5%, 6%, 7%. 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, etc., relative tothe amount of Sema6D mRNA in a nonactivated T cell or nonactivated Bcell.

In addition, the increase in Sema6D mRNA can be at least about 0.1 fold,0.2 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 2.5 fold, 3.0 fold,3.5 fold, 4.0 fold, 4.5 fold, 5.0 fold, 5.5 fold, 6.0 fold, 6.5 fold,7.0 fold, 7.5 fold, 8.0 fold, 8.5 fold, 9.0 fold, 9.5 fold, 10 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100fold, etc., relative to the amount of Sema6D mRNA in a nonactivated Tcell or nonactivated B cell.

Additionally, in methods of this invention wherein T cell activationand/or B cell activation is inhibited, such inhibition can be detectedby identifying a decrease in Sema6D protein on the surface of a T celland/or B cell and/or by identifying a decrease in mRNA encoding Sema6Dprotein in a T cell and/or B cell. Such inhibition can be detected byidentifying a decrease in Sema6D protein and/or mRNA relative to theamount of Sema6D protein and/or mRNA present in a T cell identified asan activated T cell and/or in a B cell identified as an activate B cell.Typical surface and biochemical activation markers on T cells includebut are not limited to CD69, CD25, HLA-DR, CD62L, CD154 and/or theproduction of IL-2, calcium mobilization, ZAP-70 phosphorylation, LATphosphorylation, Lck phosphorylation and c-abl kinase activation.Immunologic assays measuring T cell and/or B cell proliferation andcytotoxicity (defined as the ability to kill target cells) can also beused.

Thus, in some embodiments, the inhibition or reduction of T cellactivation or B cell activation can be a decrease in Sema6D proteinand/or mRNA of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%, 10%,12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, etc., relative to the amount of Sema6Dprotein and/or mRNA in an activated T cell or activated B cell.

In addition, the inhibition or reduction of T cell activation or B cellactivation can be a decrease of Sema6D protein and/or mRNA of at leastabout 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 2.5fold, 3.0 fold, 3.5 fold, 4.0 fold, 4.5 fold, 5.0 fold, 5.5 fold, 6.0fold, 6.5 fold, 7.0 fold, 7.5 fold, 8.0 fold, 8.5 fold, 9.0 fold, 9.5fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, 100 fold, etc., relative to the amount of Sema6D proteinand/or mRNA in an activated T cell or activated B cell.

The present invention also provides a method of monitoring T cellactivation and/or B cell activation over time, comprising detectingSema6D on the surface of a T cell and/or B cell over time and measuringchanges in the amount of Sema6D on the surface of a T cell and/or B cellover time. Additionally provided is a method of monitoring T cellactivation and/or B cell activation over time, comprising detecting mRNAencoding Sema6D in a T cell and/or B cell over time and measuringchanges in the amount of mRNA encoding Sema6D in the cell over time.Thus a baseline measurement of Sema6D protein and/or mRNA can beperformed according to methods known in the art and as described hereinand measurements of Sema6D protein and/or mRNA can be carried out at anytime interval (e.g., minutes, hours, days, etc.) and under conditionswhereby T cell activation and/or B cell activation can be modulated.Changes in the amount of Sema6D protein and/or mRNA can be detected,whereby an increase or decrease in the amount of Sema6D protein and/ormRNA can identify an increase or decrease, respectively in theactivation of a T cell and/or B cell over time.

The present invention further provides screening methods, including amethod of identifying a substance having the ability to inhibit Sema6Dactivity, comprising contacting the substance with T cells and/or Bcells expressing Sema6D under conditions whereby Sema6D activity canoccur and measuring the amount of Sema6D activity in the presence and inthe absence of the substance; whereby a decrease in Sema6D activity inthe presence of the substance as compared to the amount of Sema6Dactivity in the absence of the substance identifies a substance havingthe ability to inhibit Sema6D activity.

Further provided herein is a method of identifying a substance havingthe ability to enhance Sema6D activity, comprising contacting thesubstance with T cells and/or B cells expressing Sema6D under conditionswhereby Sema6D activity can occur and measuring the amount of Sema6Dactivity in the presence and in the absence of the substance; whereby anincrease in Sema6D activity in the presence of the substance as comparedto the amount of Sema6D activity in the absence of the substanceidentifies a substance having the ability to enhance Sema6D activity.

In the screening methods of this invention, Sema6D activity indicatesactivation of T cells, as determined by measurement of T cell activationmarkers such as CD25, CD69, CD62L, CD154, CD44, HLA-DR, IL-2 production,calcium mobilization, phosphorylation of LAT, ZAP70, lck, c-Abl etc., inresponse to a substance that can bind and activate through the Sema6Dmolecule. An example would be a fusion protein consisting of theextramembrane domain of PlexA1 coupled with the Fc portion ofimmunoglobulin as described herein.

Sema6D activity can be measured by, for example, identifying the T celland/or B cell activation status of a T cell and/or B cell in the absenceof a test substance and measuring the T cell and/or B cell activationstatus of the T cell and/or B cell in the presence of the substance,whereby an increase in T cell and/or B cell activation in the presenceof the substance identifies the substance as having the ability toenhance Sema6D activity and whereby a decrease in T cell and/or B cellactivation in the presence of the substance identifies a substancehaving the ability to inhibit Sema6D activity. The activation status ofa T cell can be measured by methods standard in the art, including butnot limited to, measuring an increase in the production and/orexpression of CD69, CD25, HLA-DR, CD62L, CD154 and/or CD44, eithersingly or in any combination, in the T cell, according to art-knownmethods. T cell activation status can also be determined by employingart-known methods for detecting cytotoxic T cell responses, T helperresponses and/or IL-2 production. The activation status of a B cell canbe measured by methods standard in the art.

In some embodiments, the screening methods of this invention can includethe step of contacting the T cells and/or B cells with a known inhibitorof Sema6D activity, such as an antibody that specifically binds a Sema6Dprotein or a fusion protein of this invention comprising theextracellular domain of a Sema6D protein and an immunoglobulin fragmentand establishing a baseline amount of T cell and/or B cell activationand then contacting the T cell and/or B cell with the substance to bescreened and identifying a change in the T cell and/or B cell activationstatus to identify a substance that either inhibits or enhances Sema6Dactivity. Typical surface and biochemical activation markers on T cellsinclude but are not limited to CD69, CD25, HLA-DR, CD62L, CD154 and/orthe production of IL-2, calcium mobilization, ZAP-70 phosphorylation,LAT phosphorylation, and Lck phosphorylation. T cell proliferation andcytotoxicity (defined as the ability to kill target cells) can also bemeasured. Sema6D activity can be measured by the methods describedherein.

In some embodiments of this invention, substances can be screened forthe ability to inhibit or enhance Sema6D activity by affecting theability of Sema6D to bind PlexA1. This inhibition or enhancement ofbinding activity can be detected by any of a variety of art-recognizedmethods for evaluating binding activity. As one example, the substanceto be tested and a PlexA1 protein or an active fragment thereof can becontacted in the presence of T cells and/or B cells having Sema6D on thesurface. The amount of binding of PlexA1 to the cells in the presence ofthe substance and the amount of binding of PlexA1 to the cells in theabsence of the substance can be determined and a decrease or increase inthe amount of binding in the presence of the substance identifies thesubstance as having the ability to inhibit or enhance binding,respectively and thus inhibit or enhance Sema6D activity, respectively.

In some embodiments, binding of the PlexA1 protein to a T cell or B cellcan be measured by attaching a detectable moiety to the PlexA1polypeptide or fragment (e.g., a fluorescence moiety, histochemicallydetectable moiety, radioactive moiety, etc.). The amount of detectablemoiety can be measured in the presence and absence of the substance tobe tested and the amounts can be compared to determine inhibition orenhancement. T cell activation can be measured by methods not limited tothe following: detection and/or quantitation of cell surface markerssuch as CD69, CD25, HLA-DR, CD62L, CD154 and/or the production of IL-2,calcium mobilization, ZAP-70 phosphorylation, LAT phosphorylation, Lckphosphorylation; NF-κB activation, MEK activation, NFAT activation, Ap-1activation; T cell proliferation and cytotoxicity (defined as theability to kill target cells).

Substances suitable for screening according to the above methods includesmall molecules, natural products, peptides, nucleic acids, etc. Sourcesfor compounds include natural product extracts, collections of syntheticcompounds, and compound libraries generated by combinatorial chemistry.Libraries of compounds are well known in the art. Small moleculelibraries can be obtained from various commercial entities, for example,SPECS and BioSPEC B.V. (Rijswijk, the Netherlands), ChembridgeCorporation (San Diego, Calif.), Comgenex USA Inc., (Princeton, N.J.),Maybridge Chemical Ltd. (Cornwall, UK), and Asinex (Moscow, Russia). Onerepresentative example is known as DIVERSet™, available from ChemBridgeCorporation, 16981 Via Tazon, Suite G, San Diego, Calif. 92127.DIVERSet™ contains between 10,000 and 50,000 drug-like, hand-synthesizedsmall molecules. The compounds are pre-selected to form a “universal”library that covers the maximum pharmacophore diversity with the minimumnumber of compounds and is suitable for either high throughput or lowerthroughput screening. For descriptions of additional libraries, see, forexample, Tan et al. “Stereoselective Synthesis of Over Two MillionCompounds Having Structural Features Both Reminiscent of NaturalProducts and Compatible with Miniaturized Cell-Based Assays” Am. ChemSoc. 120, 8565-8566, 1998; Floyd et al. Prog Med Chem 36:91-168, 1999.Numerous libraries are commercially available, e.g., from AnalytiCon USAInc., P.O. Box 5926, Kingwood, Tex. 77325; 3-DimensionalPharmaceuticals, Inc., 665 Stockton Drive, Suite 104, Exton, Pa.19341-1151; Tripos, Inc., 1699 Hanley Rd., St. Louis, Mo., 63144-2913,etc. In certain embodiments of the invention the methods are performedin a high-throughput format using techniques that are well known in theart, e.g., in multiwell plates, using robotics for sample preparationand dispensing, etc. Representative examples of various screeningmethods may be found, for example, in U.S. Pat. Nos. 5,985,829,5,726,025, 5,972,621, and 6,015,692. The skilled practitioner willreadily be able to modify and adapt these methods as appropriate.

The present invention further provides compositions, such as a fusionprotein comprising the extracellular domain of a Sema6D protein or anactive portion or fragment thereof and any active or functional fragmentof an immunoglobulin molecule, as would be well known in the art. Alsoprovided is a fusion protein comprising a transmembrane domain or anactive portion or fragment thereof of a Sema6D protein and/or anintracellular domain or an active portion or fragment thereof of aSema6D protein and an active or functional fragment of an immunoglobulinmolecule. The present invention further provides a compositioncomprising a fusion protein of this invention in a pharmaceuticallyacceptable carrier. Additionally provided is a composition comprising anantibody or other ligand that specifically binds a Sema6D protein in apharmaceutically acceptable carrier. Further provided herein is anucleotide sequence encoding a fusion protein of this invention, whichnucleotide sequence can be present in a composition comprising apharmaceutically acceptable carrier. These compositions can be deliveredto a subject of this invention in methods as described herein and inmethods of treating disorders and diseases as described hereinassociated with increased or decreased T cell and/or B cell activation.

Thus, in further embodiments, the present invention provides a method oftreating a T-cell-related disorder, B cell-related disorder and/or otherwhite blood cell related disease or disorder in a subject, comprisingadministering to the subject a therapeutic amount of an inhibitor ofSemaphorin 6D (Sema6D) activity on T cells, B cells and/or other whiteblood cells.

Nonlimiting examples of the diseases and disorders that can be treatedaccording to the methods of this invention include but are not limitedto leukemia (e.g., lymphoblastic leukemia, chronic myelogenous leukemia;promyelocytic leukemia, etc.; FIG. 1), lymphoma (e.g., B cell lymphomas,T cell lymphomas, Burkitts lymphoma, etc.), autoimmune diseases anddisorders, inflammatory disorders and diseases, transplant rejection,psoriasis, asthmatic and allergic disorders and any combination thereof.

Nonlimiting examples of autoimmune disorders and diseases that can betreated and/or prevented by the methods of this invention includearthritis (e.g., rheumatoid arthritis or RA), multiple sclerosis (MS),diabetes (e.g., insulin dependent diabetes mellitus or IDDM), systemiclupus erythematosus (SLE), allergic reactions, asthmatic reaction,myasthenia gravis, Crohns' disease, regional enteritis, vasculitis,ulcerative colitis, Sjogren's syndrome, ankylosing spondylitis,polymyositis and any other autoimmune disorder now known or lateridentified.

An inflammatory disease or disorder of this invention can include but isnot limited to inflammation of any organ, e.g., skin, heart,gastrointestinal tract, central nervous system, liver, pancreas, ovary,lung, eye, ear, throat, etc., such as, e.g., in psoriasis and generaltissue fibrosis.

Additionally provided is a method of reducing the likelihood oftransplant rejection (or increasing the likelihood of successfultransplantation) in a transplant recipient, comprising administering tothe transplant recipient an effective amount of an inhibitor of T celland/or B cell activation of this invention. The reduction in thelikelihood of transplant rejection or increase in the likelihood ofsuccessful transplantation is in comparison to the likelihood oftransplant rejection or likelihood of successful transplantation in atransplant recipient that did not receive an inhibitor of T cell and/orB cell activation, as such likelihood would be known and/or determinedaccording to art-known standards. Furthermore, the inhibitor of thesemethods can be administered to the transplant recipient at any timerelative to the transplantation (i.e., before, after and/orsimultaneously, in any combination).

In further embodiments, the present invention provides nucleic acidsthat inhibit T cell and/or B cell activation and nucleic acids thatenhance T cell and/or B cell activation. These nucleic acids can bepresent in a composition comprising a pharmaceutically acceptablecarrier. These nucleic acids can be present in vectors, plasmids, and/orother vehicles for delivery of nucleic acids to cells to carry out themethods of this invention, as described herein. These nucleic acids canencode inhibitors and enhancers of T cell and/or B cell activationand/or these nucleic acids can act directly to inhibit or enhance T celland/or B cell activation, for example, by inhibiting or enhancing Sema6Dactivity at the nucleic acid level.

Also provided herein is a method of treating a disorder or diseaseassociated with decreased T cell and/or B cell activation, comprisingadministering to the subject an effective amount of an enhancer of Tcell and/or B cell activation as described herein.

In the methods provided herein for enhancing T cell and/or B cellactivation in a subject, such an enhancement can be identified bycomparison with T cell and/or B cell activation in a subject that didnot receive the enhancer of this invention. Such comparative studies canbe carried out according to well known protocols in the art fordetecting and/or measuring T cell and/or B cell activation, and asdescribed herein.

Thus, the present invention further provides a method of initiating,inducing and/or enhancing a T cell-mediated immune response and/or a Bcell-mediated immune response in a subject, comprising administering tothe subject an effective amount of an enhancer of Semaphorin 6D (Sema6D)activity on T cells and/or B cells.

The subject of this invention can be any subject in need of theimmunomodulating effects of the methods of this invention. Such asubject can be any type of animal that is susceptible to diseases anddisorders associated with increased T cell and/or B cell activation ordecreased T cell and/or B cell activation and/or that can be treated byincreasing or decreasing T cell and/or B cell activation according tothe methods of this invention, as well as any animal to whom thecompositions of this invention can be administered according to themethods of this invention. For example, an animal of this invention canbe a mammal, a bird or a reptile. In certain embodiments, the subject ofthis invention is a human.

As noted above, the compositions of this invention can be administeredto a cell of a subject or to a subject either in vivo or ex vivo. Foradministration to a cell of the subject in vivo, as well as foradministration to the subject, the compositions of this invention can beadministered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, subcutaneousinjection, transdermally, extracorporeally, topically, by transdermalpatch, or the like.

The exact amount of the composition required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the particular composition used, its mode ofadministration, the condition being treated and the like. Thus, it isnot possible to specify an exact amount for every composition of thisinvention. However, an effective amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein.

As an example, one or more doses of between about 0.1 μg/kg and about1000 mg/kg of an inhibitor and/or biologically active fragment of thisinvention can be administered orally and/or parenterally to a subject inwhom it is desirable to decrease T cell activation, at hourly, dailyand/or weekly intervals until an evaluation of the subject's clinicalparameters indicate that the subject's condition has improved and/or thesubject demonstrates the desired response.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the subject's body according to standard protocolswell known in the art. The compositions of this invention can beintroduced into the cells via known mechanisms for uptake of materialsinto cells (e.g., phagocytosis, pulsing onto class I MHC-expressingcells, liposomes, etc.). The cells can then be infused (e.g., in apharmaceutically acceptable carrier) or transplanted back into the samesubject or a different subject per standard methods for the cell ortissue type. Standard methods are known for transplantation or infusionof various cells into a subject.

The pharmaceutical compositions of this invention include those suitablefor oral, rectal, topical, inhalation (e.g., via an aerosol) buccal(e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous,intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, intracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend, as is well known in the art, on such factors asthe species, age, gender and overall condition of the subject, thenature and severity of the condition being treated and/or on the natureof the particular composition (i.e., dosage, formulation) that is beingadministered.

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortables, each containing a predetermined amount of the composition ofthis invention; as a powder or granules; as a solution or a suspensionin an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Oral delivery can be performed by complexing acomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers include plastic capsules or tablets, as knownin the art. Such formulations are prepared by any suitable method ofpharmacy, which includes the step of bringing into association thecomposition and a suitable carrier (which may contain one or moreaccessory ingredients as noted above). In general, the pharmaceuticalcomposition according to embodiments of the present invention areprepared by uniformly and intimately admixing the composition with aliquid or finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the composition,optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the composition of thisinvention in a flavored base, usually sucrose and acacia or tragacanth;and pastilles comprising the composition in an inert base such asgelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions of this invention suitable for parenteraladministration can comprise sterile aqueous and non-aqueous injectionsolutions of the composition of this invention, which preparations arepreferably isotonic with the blood of the intended recipient. Thesepreparations can contain anti-oxidants, buffers, bacteriostats andsolutes, which render the composition isotonic with the blood of theintended recipient. Aqueous and non-aqueous sterile suspensions,solutions and emulsions can include suspending agents and thickeningagents. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The compositions can be presented in unit\dose or multi-dose containers,for example, in sealed ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, saline or water-for-injectionimmediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 0.1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration arepreferably presented as unit dose suppositories. These can be preparedby admixing the composition with one or more conventional solidcarriers, such as for example, cocoa butter and then shaping theresulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers that can be usedinclude, but are not limited to, petroleum jelly, lanoline, polyethyleneglycols, alcohols, transdermal enhancers, and combinations of two ormore thereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharmaceutical Research3:318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the composition of this invention. Suitableformulations can comprise citrate or bis\tris buffer (pH 6) orethanol/water and can contain from 0.1 to 0.2M active ingredient.

Furthermore, the compositions of this invention can be administeredorally, intranasally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like. In the methods described hereinwhich include the administration and uptake of exogenous DNA into thecells of a subject (i.e., gene transduction or transfection), thenucleic acids of the present invention can be in the form of naked. DNAor the nucleic acids can be in a vector for delivering the nucleic acidsto the cells for expression of the polypeptides and/or fragments of thisinvention. The vector can be a commercially available preparation or canbe constructed in the laboratory according to methods well known in theart.

Delivery of a nucleic acid or vector to cells can be via a variety ofmechanisms. As one example, delivery can be via a liposome, usingcommercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system, which can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells nucleic acid encoding thepolypeptide and/or fragment of this invention. The exact method ofintroducing the exogenous nucleic acid into mammalian cells is, ofcourse, not limited to the use of retroviral vectors. Other techniquesare widely available for this procedure including the use of adenoviralvectors, alphaviral vectors, adeno-associated viral (AAV) vectors,lentiviral vectors, pseudotyped retroviral vectors and vaccinia viralvectors, as well as any other viral vectors now known or developed inthe future. Physical transduction techniques can also be used, such asliposome delivery and receptor-mediated and other endocytosismechanisms. This invention can be used in conjunction with any of theseor other commonly used gene transfer methods.

As one example, if the nucleic acid of this invention is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10¹²,10¹⁵ and/or 10²⁰ pfu per injection.

In some embodiments, a subject will receive a single injection of aviral vector comprising a nucleic acid of this invention. If additionalinjections are necessary, they can be repeated at daily/weekly/monthlyintervals for an indefinite period and/or until the efficacy of thetreatment has been established. As set forth herein, the efficacy oftreatment can be determined by evaluating the symptoms and clinicalparameters described herein and/or by detecting a desired immunologicalresponse.

The exact amount of the nucleic acid or vector required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the particular nucleic acid or vector used,its mode of administration and the like. Thus, it is not possible tospecify an exact amount for every nucleic acid or vector. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein.

Further provided are isolated nucleic acids comprising, consistingessentially of and/or consisting of nucleotide sequences that encode theproteins and fragments of this invention. In particular, the presentinvention provides a fusion protein comprising, consisting essentiallyof, and/or consisting of the amino acid sequence of

(Sema6D-Ig: primary amino acid sequence (886 aa) SEQ ID NO: 2(MGFLLLWFCVLFLLVSRLRAVSFPEDDEPLNTVDYHYSRQYPVFRGRPSGNESQHRLDFQLMLKIRDTLYIAGRDQVYTVNLNEIPQTEVIPSKKLTWRSRQQDRENCAMKGKHKDECHNFIKVFVPRNDEMVFVCGTFNAFNPMCRYYRLRTLEYDGEEISGLARCPFDARQTNVALFADGKLYSATVADFLASDAVIYRSMGDGSALRTIKYDSKWIKEPHFLHAIEYGNYVYFFFREIAVEHNNLGKAVYSRVARICKNDMGGSQRVLEKHWTSFLKARLNCSVPGDSFFYFDVLQSITDIIQINGIPTVVGVFTTQLNSIPGSAVCAFSMDDIEKVFKGRFKEQKTPDSVWTAVPEDKVPKPRPGCCAKHGLAEAYKTSIDFPDDTLAFIKSHPLMDSAVPPIADEPWFTKTRVRYRLTAIEVDRSAGPYQNYTVIFVGSEAGVVLKVLAKTSPFSLNDSVLLEEIEAYNPAKCSAESEEDRKVVSLQLDKDHHALYVAFSSCVVRIPLSRCERYGSCKKSCIASRDPYCGWLSQGVCERVTLGMLPGGYEQDTEYGNTAHLGDCHDMEVSSSSVTTVASSPEITSKVIDTWRPKLTSSRKFVVQDDPNTSDFTDTISGIPKGVRWEVQSGESNQMVHMNVLITC VFAA): Sema6D seq(652 aa) (GSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK): Ig seq

Additionally provided is a nucleic acid comprising, consistingessentially of, and/or consisting of a nucleotide sequence that encodesan amino acid sequence comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof the amino acid sequence of SEQ ID NO:2 above. In a particularembodiment, the nucleic acid of this invention comprises the nucleotidesequence of

SEQ ID NO: 1: (GCCACCCATGGGGTTCC TTCTGCTTTG GTTCTGCGTG CTGTTCCTTCTGGTCTCCAG GTTACGGGCGGTCAGCTTCC CAGAAGACGA TGAGCCCCTC AACACGGTTGACTATCACTA TTCAAGGCAATATCCGGTTT TTAGAGGACG CCCTTCAGGC AACGAATCGCAGCACAGGCT GGACTTTCAGCTGATGTTGA AAATTCGAGA CACACTTTAT ATTGCTGGCAGGGATCAAGT CTATACAGTGAACTTAAATG AAATCCCCCA AACAGAGGTG ATACCAAGCAAGAAGCTGAC GTGGAGGTCCAGACAGCAGG ATCGAGAAAATTGTGCTATG AAAGGCAAGCATAAAGATGA ATGCCACAACTTCATCAAAG TCTTTGTCCC AAGAAATGAT GAGATGGTTTTTGTCTGTGG TACCAATGCTTTCAACCCGA TGTGCAGATA CTATAGGTTG AGAACGTTAGAGTATGATGG GGAAGAAATTAGTGGCCTGG CACGATGCCC GTTTGATGCC CGACAAACCAATGTCGCCCT CTTTGCTGATGGAAAACTCT ATTCTGCCAC AGTGGCTGAT TTCCTGGCCAGTGATGCTGT CATTTACAGAAGCATGGGAG ATGGATCTGC CCTTCGCACA ATAAAATACGATTCCAAGTG GATCAAAGAACCACACTTCC TTCATGCCAT AGAATATGGA AACTATGTCTATTTCTTCTT CAGAGAAATCGCCGTGGAAC ATAATAACTT AGGCAAGGCT GTGTATTCCCGCGTGGCTCG CATTTGTAAAAACGACATGG GTGGCTCACA GCGGGTCCTG GAGAAACACTGGACTTCCTT CCTTAAGGCTCGGCTGAACT GCTCCGTTCC TGGAGATTCC TTTTTCTACTTCGACGTCCT GCAGTCTATAACAGACATAA TCCAAATCAATGGCATCCCC ACTGTGGTTGGGGTCTTCAC CACACAGCTCAACAGCATTCCTGGTTCTGC AGTCTGTGCC TTTAGCATGGACGACATTGAGAAAGTGTTCAAAGGGCGGT TCAAAGAGCA GAAAACCCCA GACTCTGTTTGGACAGCAGT TCCCGAAGACAAAGTACCAA AACCAAGGCC TGGCTGTTGT GCCAAACACGGCCTCGCAGA AGCTTACAAGACCTCCATCG ACTTTCCAGA TGACACCCTG GCTTTCATCAAGTCCCACCC GCTGATGGACTCTGCCGTCC CACCCATTGC CGATGAGCCC TGGTTCACAAAGACACGGGT CAGGTACAGGTTGACAGCCA TCGAAGTGGA CCGTTCAGCA GGGCCATACCAAAACTACAC AGTCATCTTTGTTGGCTCTG AAGCTGGCGT GGTACTTAAA GTTTTGGCAAAGACCAGTCC TTTCTCTCTGAATGACAGTG TATTACTCGA AGAGATTGAA GCTTATAACCCAGCCAAGTG CAGCGCCGAGAGTGAGGAGG ACAGAAAGGT GGTCTCATTA CAGCTGGACAAGGATCACCA TGCTTTATACGTGGCCTTCT CTAGCTGCGT GGTCCGCATC CCCCTCAGCCGCTGTGAGCG CTACGGATCGTGTAAAAAGT CTTGCATTGC ATCACGTGAC CCGTACTGTGGTTGGTTAAG CCAGGGAGTTTGTGAGAGAG TGACCCTAGG GATGCTCCCT GGAGGATATGAGCAGGACACGGAGTACGGCAACACAGCCC ACCTAGGGGA CTGCCACGAC ATGGAGGTATCCTCATCTTC TGTTACCACTGTGGCAAGTA GCCCAGAAAT TACATCTAAA GTGATTGATACCTGGAGACC TAAACTGACGAGCTCCCGGA AATTTGTAGT TCAAGATGAC CCAAATACTTCTGATTTTAC TGATACTATATCAGGTATCC CAAAGGGTGT ACGGTGGGAA GTCCAGTCTGGAGAATCCAA TCAGATGGTCCACATGAATG TCCTCATCAC CTGCGTGTTT GCCGCTGGATCCGAGCCCAA ATCTTGTGACA AAACTCACAC ATGCCCACCG TGCCCAGCAC CTGAACTCCTGGGGGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCGGACCCCTGAG GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TACACCCTGCCCCCATCCCGGGATGAGCTG ACCAAGAACC AGGTCAGCCT GACCTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGTAAATGA).

Further provided herein is a nucleic acid that is the complement of eachand any of the nucleic acids of this invention.

A variety of protocols for detecting the presence of and/or measuringthe amount of Sema6D protein, using, e.g., polyclonal and/or monoclonalantibodies specific for the Sema6D protein, are known in the art.Examples of such protocols include, but are not limited to, enzymeimmunoassays (EIA), agglutination assays, immunoblots (Western blot;dot/slot blot, etc.), radioimmunoassays (RIA), immunodiffusion assays,chemiluminescence assays, antibody library screens, expression arrays,enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA),immunoprecipitation, Western blotting, competitive binding assays,immunofluorescence, immunohistochemical stainingprecipitation/flocculation assays and fluorescence-activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton et al. (Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn. (1990)) and Maddox et al. (J. Exp. Med.158:1211-1216 (1993)).

Furthermore, a number of assays for identification, detection and/oramplification of nucleic acid sequences (e.g., Sema6D mRNA) are wellknown in the art. For example, various protocols can be employed in themethods of this invention to amplify nucleic acid. As used herein, theterm “oligonucleotide-directed amplification procedure” refers totemplate-dependent processes that result in an increase in theconcentration of a specific nucleic acid molecule relative to itsinitial concentration, or in an increase in the concentration of adetectable signal, such as amplification. As used herein, the term“oligonucleotide directed mutagenesis procedure” is intended to refer toa process that involves the template-dependent extension of a primermolecule. The term “template dependent process” refers to nucleic acidsynthesis of a RNA or a DNA molecule wherein the sequence of the newlysynthesized strand of nucleic acid is dictated by the well-known rulesof complementary base pairing. Typically, vector mediated methodologiesinvolve the introduction of the nucleic acid fragment into a DNA or RNAvector, the clonal amplification of the vector, and the recovery of theamplified nucleic acid fragment. Examples of such methodologies areprovided in U.S. Pat. No. 4,237,224 (incorporated herein by reference inits entirety). Nucleic acids, used as a template for amplificationmethods can be isolated from cells according to standard methodologies(Sambrook et al., 1989). The nucleic acid can be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary DNA. In one embodiment, the RNA canbe whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to the Sema6D gene or coding sequence are contacted withthe nucleic acid under conditions that permit selective hybridization.The term “primer,” as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template dependent process. Typically, primers are oligonucleotidesfrom ten to twenty bases in length, but shorter (e.g., 6, 7, 8, or 9bases) or longer (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50 bases) sequences can be employed. Primers can in double-strandedor single-stranded form, although the single-stranded form is commonlyused.

Once hybridized, the nucleic acid: primer hybridization complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In some embodiments, thedetection can be performed by visual means. Alternatively, the detectioncan involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescence or chemiluminescence label or even via a system usingelectrical or thermal impulse signals (e.g., Affymax technology).

A number of template dependent processes are available to amplify thesequences present in a given template sample. One of the best-knownamplification methods is the polymerase chain reaction (referred to asPCR), which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, each incorporated herein by reference in itsentirety.

Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase, e.g., a Taq polymerase. Ifthe particular target sequence is present in a sample, the primers willbind to the target sequence and the polymerase will cause the primers tobe extended along the sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the target sequence to form reaction products,excess primers will bind to the target sequence and to the reactionproducts and the process is repeated.

A reverse transcriptase PCR amplification procedure can be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known in the art (e.g., Sambrook etal., 1989). Alternative methods for reverse transcription employthermostable, RNA-dependent DNA polymerases. These methods aredescribed, for example, in PCT Publication No. WO 90/07641, filed Dec.21, 1990, incorporated herein by reference in its entirety. Polymerasechain reaction methodologies are well known in the art.

Another method for nucleic acid amplification is the ligase chainreaction (“LCR”), disclosed in Eur. Pat. Appl. No. 320308, incorporatedherein by reference in its entirety. In LCR, two complementary probepairs are prepared and in the presence of the target sequence, each pairwill bind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750 (incorporated byreference herein in its entirety) describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta replicase (QβR), described in PCT Application No. PCT/US87/00880,(incorporated herein by reference), can also be used as an amplificationmethod in the present invention. In this method, a replicative sequenceof RNA that has a region complementary to that of a target is added to asample in the presence of an RNA polymerase. The polymerase will copythe replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA), described in U.S. Pat. Nos.5,455,166, 5,648,211, 5,712,124 and 5,744,311, each incorporated hereinby reference, is another method of carrying out isothermal amplificationof nucleic acids which involves multiple rounds of strand displacementand synthesis, i.e., nick translation. A similar method, called RepairChain Reaction (RCR), involves annealing several probes throughout aregion targeted for amplification, followed by a repair reaction inwhich only two of the four bases are present.

The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification method, as described in Intl. Pat. Appl. No.PCT/US89/01025, which is incorporated herein by reference in itsentirety, may be used in accordance with the present invention. In oneembodiment, “modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detectable moiety (e.g.,enzyme). In another embodiment, an excess of labeled probes is added toa sample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact, available to be bound by excess probe. Cleavage of the labeledprobe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (PCT Publication No. WO 88/10315,incorporated herein by reference). In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer that has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7, T3 orSP6. In an isothermal cyclic reaction, the RNAs are reverse transcribedinto single stranded DNA, which is then converted to double-strandedDNA, and then transcribed once again with an RNA polymerase such as T7,T3 or SP6. The resulting products, whether truncated or complete,indicate target specific sequences.

European Pat. Appl. No. 329822 (incorporated herein by reference in itsentirety) discloses a nucleic acid amplification process involvingcyclically synthesizing single stranded RNA (ssRNA), ssDNA, anddouble-stranded DNA (dsDNA), which can be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA).

The resultant ssDNA is a template for a second primer, which alsoincludes the sequences of an RNA polymerase promoter (exemplified by T7RNA polymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large Klenow fragment ofE. coli DNA polymerase I), resulting in a double-stranded DNA (dsDNA)molecule, having a sequence identical to that of the original RNAbetween the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle, leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) discloses a nucleic acid sequence amplification scheme basedon the hybridization of a promoter/primer sequence to a targetsingle-stranded DNA (ssDNA), followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporatedby reference herein).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the dioligonucleotide, can alsobe used in the amplification step of the present invention.

Following any amplification, it is desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products can be separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (e.g., Sambrook et al., 1989).

Alternatively, chromatographic techniques can be used to effectseparation. There are many kinds of chromatography that can be used inthe present invention: such as, for example, adsorption, partition, ionexchange and molecular sieve, as well as many specialized techniques forusing them including column, paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the target sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In some embodiments, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified target sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In other embodiments, detection can be by Southern or Northern blottingand hybridization with a labeled probe. The techniques involved inSouthern and Northern blotting are well known to those of skill in theart and can be found in many standard books on molecular protocols(e.g., Sambrook et al., 1989). Briefly, amplification products areseparated by gel electrophoresis. The gel is then contacted with amembrane, such as nitrocellulose, permitting transfer of the nucleicacid and noncovalent binding. Subsequently, the membrane is incubatedwith a chromophore-conjugated probe that is capable of hybridizing witha target amplification product. Detection is by exposure of the membraneto x-ray film or ion-emitting detection devices. One example of theforegoing is described in U.S. Pat. No. 5,279,721, incorporated byreference herein, which discloses an apparatus and method for theautomated electrophoresis and transfer of nucleic acids. The apparatuspermits electrophoresis and blotting without external manipulation ofthe gel.

Additionally, a wide variety of labeling and conjugation techniques areknown in the art that are used in various nucleic acid detection andamplification assays. Methods for producing labeled hybridization probesand/or PCR or other ligation primers for detecting and/or amplifyingnucleic acid sequences can include, for example, oligolabeling, nicktranslation and end-labeling, as well as other well known methods.Alternatively, nucleic acid sequences encoding the polypeptides of thisinvention, and/or any functional fragment thereof, can be cloned into aplasmid or vector for detection and amplification. Such plasmids andvectors are well known in the art and are commercially available. It isalso contemplated that the methods of this invention can be conductedusing a variety of commercially available kits (e.g., Pharmacia &Upjohn; Promega; U.S. Biochemical Corp.). Suitable reporter molecules orlabels, which can be used for ease of detection, include, for example,radionuclides, enzymes, fluorescence agents, chemiluminescence agentsand chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles and the like as are well known in the art.

The present invention further includes isolated polypeptides, peptides,proteins, fragments, domains and/or nucleic acid molecules that aresubstantially equivalent to those described for this invention. As usedherein, “substantially equivalent” can refer both to nucleic acid andamino acid sequences, for example a mutant sequence, that varies from areference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an undesirableadverse functional dissimilarity between reference and subjectsequences. In some embodiments, this invention can include substantiallyequivalent sequences that have an adverse functional dissimilarity. Forpurposes of the present invention, sequences having equivalentbiological activity and equivalent expression characteristics areconsidered substantially equivalent.

The invention further provides homologs, as well as methods of obtaininghomologs, of the polypeptides and/or fragments of this invention. Asused herein, an amino acid sequence or protein is defined as a homologof a polypeptide or fragment of the present invention if it sharessignificant homology to one of the polypeptides and/or fragments of thepresent invention. Significant homology means at least 60%, 65%, 75%,80%, 85%, 90%, 95%, 98% and/or 100% homology with another amino acidsequence. Specifically, by using the nucleic acids disclosed herein as aprobe or as primers, and techniques such as PCR amplification andcolony/plaque hybridization, one skilled in the art can identifyhomologs of the polypeptides and/or fragments of this invention.

In further embodiments, the nucleic acids encoding the polypeptidesand/or fragments of this invention can be part of a recombinant nucleicacid construct comprising any combination of restriction sites and/orfunctional elements as are well known in the art that facilitatemolecular cloning and other recombinant DNA manipulations. Thus, thepresent invention further provides a recombinant nucleic acid constructcomprising a nucleic acid encoding a polypeptide and/or biologicallyactive fragment of this invention.

The present invention further provides a vector comprising a nucleicacid encoding a polypeptide and/or fragment of this invention. Thevector can be an expression vector which contains all of the geneticcomponents required for expression of the nucleic acid in cells intowhich the vector has been introduced, as are well known in the art. Theexpression vector can be a commercial expression vector or it can beconstructed in the laboratory according to standard molecular biologyprotocols. The expression vector can comprise viral nucleic acidincluding, but not limited to, poxvirus, vaccinia virus, adenovirus,retrovirus and/or adeno-associated virus nucleic acid. The nucleic acidor vector of this invention can also be in a liposome or a deliveryvehicle, which can be taken up by a cell via receptor-mediated or othertype of endocytosis.

The nucleic acid of this invention can be in a cell, which can be a cellexpressing the nucleic acid whereby a polypeptide and/or biologicallyactive fragment of this invention is produced in the cell. In addition,the vector of this invention can be in a cell, which can be a cellexpressing the nucleic acid of the vector whereby a polypeptide and/orbiologically active fragment of this invention is produced in the cell.It is also contemplated that the nucleic acids and/or vectors of thisinvention can be present in a host animal (e.g., a transgenic animal),which expresses the nucleic acids of this invention and produces thepolypeptides and/or fragments of this invention.

The nucleic acid encoding the polypeptide and/or fragment of thisinvention can be any nucleic acid that functionally encodes thepolypeptides and/or fragments of this invention. To functionally encodethe polypeptides and/or fragments (i.e., allow the nucleic acids to beexpressed), the nucleic acid of this invention can include, for example,expression control sequences, such as an origin of replication, apromoter, an enhancer and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences.

Nonlimiting examples of expression control sequences that can be presentin a nucleic acid of this invention include promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected polypeptide and/or fragment can readily be determined basedupon the genetic code for the amino acid sequence of the selectedpolypeptide and/or fragment and many nucleic acids will encode anyselected polypeptide and/or fragment. Modifications in the nucleic acidsequence encoding the polypeptide and/or fragment are also contemplated.Modifications that can be useful are modifications to the sequencescontrolling expression of the polypeptide and/or fragment to makeproduction of the polypeptide and/or fragment inducible or repressibleas controlled by the appropriate inducer or repressor. Such methods arestandard in the art. The nucleic acid of this invention can be generatedby means standard in the art, such as by recombinant nucleic acidtechniques and/or by synthetic nucleic acid synthesis or in vitroenzymatic synthesis.

The nucleic acids and/or vectors of this invention can be transferredinto a host cell (e.g., a prokaryotic or eukaryotic cell) by well-knownmethods, which vary depending on the type of cell host. For example,calcium chloride transfection is commonly used for prokaryotic cells,whereas calcium phosphate treatment, transduction and/or electroporationcan be used for other cell hosts.

As used herein, “a” or “an” or “the” can mean one or more than one. Forexample, “a” cell can mean one cell or a plurality of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

A T cell of this invention includes but is not limited to CD4+ T cells,T regulatory cells, double positive (CD4+, CD8+) T cells and doublenegative (CD4−, CD8−) T cells. A B cell of this invention is, e.g., anantibody producing cell and can be for example, a plasma B cell, amemory B cell, a B-1 cell or a B-2 cell.

As used herein, “T cell activation” or “B cell activation” means aprocess or activity that causes T cells or B cells to exhibit aphenotype of an activated T cell or B cell, and “activated T cell” or“activated B cell” describes T cells or B cells that can exhibit some ofthe following phenotypes: T cell activation can be measured by methodsnot limited to the following: CD69, CD25, HLA-DR, CD62L and/or CD154expression and/or the production of IL-2, calcium mobilization, ZAP-70phosphorylation, LAT phosphorylation, Lck phosphorylation, NF-κBactivation, MEK activation, NFAT activation, Ap-1 activation; T cellproliferation and cytotoxicity (defined as the ability to kill targetcells). B cell activation can be measured by any methods known in theart to identify antigen-mediated activation, T cell dependentactivation, T cell-independent activation, etc.

Nonlimiting examples of a Sema6D protein of this invention have an aminoacid sequence as shown in the Sequence Listing. For example SEQ IDNOs:22, 24, 26, 28, 30 and are examples of human isoforms of a Sema6Dprotein. Other Sema6D proteins as are known in the art and as describedherein are also included in the present invention.

As used herein, “modulate,” “modulates” or “modulation” refers toenhancement (e.g., an increase) or inhibition (e.g., diminished, reducedor suppressed) of the specified activity. The term “enhancement,”“enhance,” “enhances,” or “enhancing” refers to an increase in thespecified parameter (e.g., at least about a 1.1-fold, 1.25-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold,twelve-fold, or even fifteen-fold or more increase) and/or an increasein the specified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%,60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%. The term “inhibit,”“diminish,” “reduce” or “suppress” refers to a decrease in the specifiedparameter (e.g., at least about a 1.1-fold, 1.25-fold, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or evenfifteen-fold or more decrease) and/or a decrease or reduction in thespecified activity of at least about 5%, 10%, 25%, 35%, 40%, 50%, 60%,75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%. In particular embodiments,the inhibition or reduction results in little or essentially nodetectable activity (at most, an insignificant amount, e.g., less thanabout 10% or about 5%).

The term “overexpress,” “overexpresses” or “overexpression” as usedherein in connection with isolated nucleic acids encoding Sema6D refersto expression that results in higher levels of Sema6D polypeptide thanexist in the cell in its native (control) state. Overexpression ofSema6D can result in levels that are 25%, 50%, 100%, 200%, 500%, 1000%,2000% or higher in the cell. Further, nucleic acid encoding Sema6D canbe introduced into a cell that does not produce the specified form ofSema6D (e.g., an isoform) encoded by the transgene or does so only atnegligible levels.

The term “enhance,” “enhances,” “enhancing” or “enhancement” withrespect to T cell or B cell activation refers to an increase in T cellor B cell activation (e.g., at least about a 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or evenfifteen-fold or more increase), for example, in response to a substancethat enhances T cell or B cell activation. Alternatively, these termscan refer to increasing expression of nucleic acid encoding Sema6D in acell or subject in response to an enhancer as compared with the amountof Sema6D nucleic acid expression in the absence of the enhancer.

A “fusion polypeptide” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame. Illustrative fusionpolypeptides include, but are not limited to a fusion of theextracellular domain of Sema6D or active fragment thereof to animmunoglobulin fragment as described herein. Ig fragments from human,mouse, rat, goat, rabbit can all be used. In addition, mutations in theFc binding sequence do not alter the function of the protein, and thesecan also be used. When used in animals, it is best to use the IgG fusionthat is from the same species. For example, using human IgG fusionprotein to perform in mice may cause immunogenicity in the long run,although for short term experiments, this is less of a concern.

As used herein, a “functional” or “active” polypeptide is one thatretains at least one biological activity normally associated with thatpolypeptide. Preferably, a “functional” polypeptide retains all of theactivities possessed by the unmodified peptide. By “retains” biologicalactivity, it is meant that the polypeptide retains at least about 50%,60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biologicalactivity of the native polypeptide (and can even have a higher level ofactivity than the native polypeptide). A “non-functional” polypeptide isone that exhibits essentially no detectable biological activity normallyassociated with the polypeptide (e.g., at most, only an insignificantamount, e.g., less than about 10% or even 5%).

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim, “and those that donot materially affect the basic and novel characteristic(s)” of theclaimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03.Thus, the term “consisting essentially of” when used in a claim of thisinvention is not intended to be interpreted to be equivalent to“comprising.”

“Isolated” as used herein means the nucleic acid or protein or proteinfragment of this invention is sufficiently free of contaminants or cellcomponents with which nucleic acids or proteins normally occur.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to provide the nucleic acidor protein or protein fragment in a form in which it can be usedtherapeutically.

“Epitope” or “antigenic epitope” or “antigenic peptide” as used hereinmeans a specific amino acid sequence which, when present in the properconformation, provides a reactive site for an antibody or T cellreceptor. The identification of epitopes on antigens can be carried outby immunology protocols that are well known in the art. Typically, anepitope or antigenic peptide can be 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,18, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

As used herein, the term “polypeptide” or “protein” is used to describea chain of amino acids that correspond to those encoded by a nucleicacid. A polypeptide of this invention can be a peptide, which usuallydescribes a chain of amino acids of from two to about 30 amino acids.The term polypeptide as used herein also describes a chain of aminoacids having more than 30 amino acids and can be a fragment or domain ofa protein or a full length protein. Furthermore, as used herein, theterm polypeptide can refer to a linear chain of amino acids or it canrefer to a chain of amino acids that has been processed and folded intoa functional protein. It is understood, however, that 30 is an arbitrarynumber with regard to distinguishing peptides and polypeptides and theterms can be used interchangeably for a chain of amino acids. Thepolypeptides of the present invention are obtained by isolation andpurification of the polypeptides from cells where they are producednaturally, by enzymatic (e.g., proteolytic) cleavage, and/orrecombinantly by expression of nucleic acid encoding the polypeptides orfragments of this invention. The polypeptides and/or fragments of thisinvention can also be obtained by chemical synthesis or other knownprotocols for producing polypeptides and fragments.

The amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. Nucleotide sequences arepresented herein in the 5′ to 3′ direction, from left to right. It isintended that the nucleic acids of this invention can be either singleor double stranded (i.e., including the complementary nucleic acid). Anucleic acid of this invention can be the complement of a nucleic aciddescribed herein.

A “biologically active fragment” or “active fragment” or “functionalfragment” or “functionally active fragment” as used herein includes apolypeptide of this invention that comprises a sufficient number ofamino acids to have one or more of the biological activities of thepolypeptides of this invention. Such biological activities can include,but are not limited to, in any combination, binding activity,immunomodulating activity and/or immunogenic activity, as well as anyother activity now known or later identified for the polypeptides and/orfragments of this invention. A fragment of a polypeptide of thisinvention can be produced by methods well known and routine in the art.Fragments of this invention can be produced, for example, by enzymaticor other cleavage of naturally occurring peptides or polypeptides or bysynthetic protocols that are well known. Such fragments can be testedfor one or more of the biological activities of this invention accordingto the methods described herein, which are routine methods for testingactivities of polypeptides, and/or according to any art-known androutine methods for identifying such activities. Such production andtesting to identify biologically active fragments of the polypeptidesdescribed herein would be well within the scope of one of ordinary skillin the art and would be routine.

Fragments of the polypeptides of this invention are preferably at leastabout ten amino acids in length and retain one or more of the biologicalactivities (e.g., immunomodulating; binding) and/or the immunologicalactivities of the proteins of this invention. Examples of the fragmentsof this invention include, but are not intended to be limited to, thefollowing fragments identified by the amino acid number as shown in theSequence Listing herein: Amino acids 1-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 110-120, 120-130, 130-140, 140-150,150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220, 220-230,230-240, 240-250, 1-25, 1-50, 1-67, 1-75, 1-100, 1-125, 1-135, 1-145,1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-250, 68-180, 183-223,50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-650, etc.

It is understood that this list is exemplary only and that a fragment ofthis invention can be any amino acid sequence containing any combinationof contiguous amino acids that are numbered in the Sequence Listing asamino acids 1 through 652, even if that combination is not specificallyrecited as an example herein. It is also understood that these fragmentscan be combined in any order or amount. For example, fragment 1-10 canbe combined with fragment 10-20 to produce a fragment of amino acids1-20. As another example, fragment 1-20 can be combined with fragment50-60 to produce a single fragment of this invention having 31 aminoacids (AA 10-20 and AA 50-60). Also fragments can be present in multiplenumbers and in any combination in a fragment of this invention. Thus,for example, fragment 1-150 can be combined with a second fragment 1-150and/or combined with fragment 400-500 to produce a fragment of thisinvention.

The terms “homology,” “identity” and “complementarity” as used hereinrefer to a degree of similarity between two or more sequences. There maybe partial homology or complete homology (i.e., identity). A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence can be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of low stringency, as this term isknown in the art. This is not to say that conditions of low stringencyare such that non-specific binding is permitted; low stringencyconditions require that the binding of two sequences to one another be aspecific (i.e., selective) interaction. The absence of non-specificbinding can be tested by the use of a second target sequence that lackseven a partial degree of complementarity (e.g., less than about 30%identity). In the absence of non-specific binding, the probe will nothybridize to the second non-complementary target sequence.

The term “hybridization” as used herein refers to any process by which afirst strand of nucleic acid binds with a second strand of nucleic acidthrough base pairing. Nucleic acids encoding the polypeptides and/orfragments of this invention can be detected by DNA-DNA or DNA-RNAhybridization and/or amplification using probes, primers and/orfragments of polynucleotides encoding the polypeptides and/or fragmentsof this invention and/or designed to detect and/or amplify the nucleicacids of this invention.

The term “hybridization complex” as used herein refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells and/or nucleic acids have beenfixed).

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid,”“oligonucleotide” and “polynucleotide” are also used interchangeablyherein to refer to a heteropolymer of nucleotides. Generally, nucleicacid segments provided by this invention may be assembled from fragmentsof the genome and short oligonucleotide linkers, or from a series ofoligonucleotides, or from individual nucleotides, to provide a syntheticnucleic acid which is capable of being expressed in a recombinanttranscriptional unit comprising regulatory elements derived from amicrobial or viral operon, or a eukaryotic gene. Nucleic acids of thisinvention can comprise a nucleotide sequence that can be identical insequence to the sequence which is naturally occurring or, due to thewell-characterized degeneracy of the nucleic acid code, can includealternative codons that encode the same amino acid as that which isfound in the naturally occurring sequence. Furthermore, nucleic acids ofthis invention can comprise nucleotide sequences that can include codonswhich represent conservative substitutions of amino acids as are wellknown in the art, such that the biological activity of the resultingpolypeptide and/or fragment is retained.

The term “probe” or “primer” includes naturally occurring and/orrecombinant and/or chemically synthesized single- and/or double-strandednucleic acids. They can be labeled for detection by nick translation,Klenow fill-in reaction, PCR and/or other methods well known in the art.Probes and primers of the present invention, their preparation and/orlabeling are described in Sambrook et al. 1989. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al.1989. Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y., both of which are incorporated herein by reference in theirentirety for these teachings.

The term “stringent” as used herein refers to hybridization conditionsthat are commonly understood in the art to define the conditions of thehybridization procedure. Stringency conditions can be low, high ormedium, as those terms are commonly know in the art and well recognizedby one of ordinary skill. In various embodiments, stringent conditionscan include, for example, highly stringent (i.e., high stringency)conditions (e.g., hybridization in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS atabout 68° C.), and/or moderately stringent (i.e., medium stringency)conditions (e.g., washing in 0.2×SSC/0.1% SDS at about 42° C.).

“Amplification” as used herein includes the production of multiplecopies of a nucleic acid molecule and is generally carried out usingpolymerase chain reaction (PCR) and/or any other amplificationtechnologies as are well known in the art (Dieffenbach and Dveksler.1995. PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.).

“Effective amount” as used herein refers to an amount of a compound,agent, substance or composition of this invention that is sufficient toproduce a desired effect, which can be a therapeutic effect. Theeffective amount will vary with the age, general condition of thesubject, the severity of the condition being treated, the particularcompound, agent, substance or composition administered, the duration ofthe treatment, the nature of any concurrent treatment, thepharmaceutically acceptable carrier used if any, and like factors withinthe knowledge and expertise of those skilled in the art. As appropriate,an “effective amount” in any individual case can be determined by one ofordinary skill in the art by reference to the pertinent texts andliterature and/or by using routine experimentation. (Remington, TheScience And Practice of Pharmacy (20th ed. 2000)).

A “pharmaceutically acceptable” component such as a salt, carrier,excipient or diluent of a composition according to the present inventionis a component that (i) is compatible with the other ingredients of thecomposition in that it can be combined with the compositions of thepresent invention without rendering the composition unsuitable for itsintended purpose, and (ii) is suitable for use with subjects as providedherein without undue adverse side effects (such as toxicity, irritation,and allergic response). Side effects are “undue” when their riskoutweighs the benefit provided by the composition. Non-limiting examplesof pharmaceutically acceptable components (e.g., pharmaceuticallyacceptable carriers) include any of the standard pharmaceutical carrierssuch as phosphate buffered saline solutions, water, emulsions such asoil/water emulsion, microemulsions and various types of wetting agents.In particular, it is intended that a pharmaceutically acceptable carrierbe a sterile carrier that is formulated for administration to ordelivery into a subject of this invention.

The compositions of the present invention can also include othermedicinal agents, pharmaceutical agents, carriers, diluents,immunostimulatory cytokines, etc. and can be in a pharmaceuticallyacceptable carrier. Actual methods of preparing such dosage forms areknown, or will be apparent, to those skilled in this art.

An “immunomodulatory molecule” of this invention can be, but is notlimited to an immunostimulatory cytokine that can be, but is not limitedto, GM/CSF, interleukin-2, interleukin-12, interferon-gamma,interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoieticfactor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2co-stimulatory molecules.

Additional examples of an immunomodulatory molecule of this inventioninclude the adjuvants of this invention, including, for example, SYNTEXadjuvant formulation I (SAF-1) composed of 5 percent (wt/vol) squalene(DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (AldrichChemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) inphosphate-buffered saline. Suitable adjuvants also include an aluminumsalt such as aluminum hydroxide gel (alum), aluminum phosphate, oralgannmulin, but may also be a salt of calcium, iron or zinc, or may bean insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatized polysaccharides, orpolyphosphazenes.

Other adjuvants are well known in the art and include QS-21, Freund'sadjuvant (complete and incomplete), aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

Additional adjuvants can include, for example, a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA (3D-MPL) together with an aluminum salt. An enhanced adjuvant systeminvolves the combination of a monophosphoryl lipid A and a saponinderivative, particularly the combination of QS21 and 3D-MPL as disclosedin PCT publication number WO 94/00153 (the entire contents of which areincorporated herein by reference), or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in PCTpublication number WO 96/33739 (the entire contents of which areincorporated herein by reference). A particularly potent adjuvantformulation involving QS21 3D-MPL & tocopherol in an oil in wateremulsion is described in PCT publication number WO 95/17210 (the entirecontents of which are incorporated herein by reference). In addition,the nucleic acid of this invention can include an adjuvant by comprisinga nucleotide sequence encoding a Sema6D protein or active fragmentthereof of this invention and a nucleotide sequence that provides anadjuvant function, such as CpG sequences. Such CpG sequences, or motifs,are well known in the art. Other TLR agonists, such as Pam3Cys,Poly(I:C), single stranded RNA, as well as CATERPILLER (NOD-LRR)agonists, such as proteoglycan-derived products, are also includedherein.

The terms “treat,” “treating” or “treatment” include any type of actionthat imparts a modulating effect, which, for example, can be abeneficial effect, to a subject afflicted with a disorder, disease,condition or illness, including improvement in the disorder, disease,condition or illness of the subject (e.g., in one or more symptoms),delay in the progression of the disorder, disease, condition or illness,prevention or delay of the onset of the disorder, disease, condition orillness, and/or change in clinical parameters, disorder, disease,condition or illness status, etc., as would be well known in the art.

As used herein, the term “antibody” includes intact immunoglobulinmolecules as well as fragments thereof that are capable of binding theepitopic determinant of an antigen (i.e., antigenic determinant).Antibodies that bind the polypeptides of this invention are preparedusing intact polypeptides or fragments as the immunizing antigen. Thepolypeptide or fragment used to immunize an animal can be derived fromenzymatic cleavage, recombinant expression, isolation from biologicalmaterials, synthesis, etc., and can be conjugated to a carrier protein,if desired. Commonly used carriers that are chemically coupled topeptides and proteins for the production of antibody include, but arenot limited to, bovine serum albumin, thyroglobulin and keyhole limpethemocyanin. The coupled peptide or protein is then used to immunize theanimal (e.g., a mouse, rat, or rabbit). The polypeptide or peptideantigens can also be administered with an adjuvant, as described hereinand as otherwise known in the art.

An antibody of this invention can be any type of immunoglobulin,including IgG, IgM, IgA, IgD, and/or IgE. The antibody can be monoclonalor polyclonal and can be of any species of origin, including, forexample, mouse, rat, rabbit, horse, goat, sheep or human, or can be achimeric or humanized antibody (e.g., Walker et al., Molec. Immunol.26:403-11 (1989)). The antibodies can be recombinant monoclonalantibodies produced according to the methods disclosed in U.S. Pat. No.4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also bechemically constructed according to methods disclosed in U.S. Pat. No.4,676,980. The antibody can further be a single chain antibody (e.g.,scFv) or bispecific antibody.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science254:1275-1281). Antibodies can also be obtained by phage displaytechniques known in the art or by immunizing a heterologous host with acell containing an epitope of interest.

The polypeptide, fragment or antigenic epitope that is used as animmunogen can be modified or administered in an adjuvant in order toincrease antigenicity. Methods of increasing the antigenicity of aprotein or peptide are well known in the art and include, but are notlimited to, coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For example, for the production of antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others, can be immunized byinjection with the polypeptides and/or fragments of this invention, withor without a carrier protein. Additionally, various adjuvants may beused to increase the immunological response. Such adjuvants include, butare not limited to, Freund's complete and incomplete adjuvants, mineralgels such as aluminum hydroxide, and surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areespecially preferable.

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein (Nature 265:495-97 (1975)).Other techniques for the production of monoclonal antibodies include,but are not limited to, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kozbor et al. 1985. J. Immunol. Methods81:31-42; Cote et al. 1983. Proc. Natl. Acad. Sci. 80:2026-2030; Cole etal. 1984. Mol. Cell. Biol. 62:109-120).

For example, to produce monoclonal antibodies, a solution containing theappropriate antigen can be injected into a mouse and, after a sufficienttime, the mouse sacrificed and spleen cells obtained. The spleen cellsare then immortalized by fusing them with myeloma cells or with lymphomacells, typically in the presence of polyethylene glycol, to producehybridoma cells. The hybridoma cells are then grown in a suitable mediumand the supernatant screened for monoclonal antibodies having thedesired specificity. Monoclonal Fab fragments can be produced in abacterial cell such as E. coli by recombinant techniques known to thoseskilled in the art (e.g., Huse. Science 246:1275-81 (1989)). Any one ofa number of methods well known in the art can be used to identify thehybridoma cell, which produces an antibody with the desiredcharacteristics. These include screening the hybridomas by ELISA assay,Western blot analysis, or radioimmunoassay. Hybridomas secreting thedesired antibodies are cloned and the class and subclass are identifiedusing standard procedures known in the art.

For polyclonal antibodies, antibody-containing serum is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using any of the well known procedures asdescribed herein.

The present invention further provides antibodies of this invention indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescence labels (such as FITC or rhodamine,etc.), paramagnetic atoms, gold beads, etc. Such labeling procedures arewell-known in the art. The labeled antibodies of the present inventioncan be used for in vitro, in vivo, and in situ assays to identify apolypeptide and/or fragment of this invention in a sample.

In some embodiments, the present invention further provides theabove-described antibodies immobilized on a solid support (e.g., beads,plates, slides or wells formed from materials such as latex orpolystyrene). Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., Handbook of Experimental Immunology 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10 (1986)). Antibodiescan likewise be conjugated to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase,alkaline phosphatase), and fluorescence labels (e.g., fluorescein) inaccordance with known techniques. Determination of the formation of anantibody/antigen complex in the methods of this invention can be bydetection of, for example, precipitation, agglutination, flocculation,radioactivity, color development or change, fluorescence, luminescence,etc., as is well know in the art.

In addition, techniques developed for the production of chimericantibodies or humanized antibodies by splicing mouse antibody genes tohuman antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity can be used (Morrison et al. 1984.Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. 1984. Nature312:604-608; Takeda et al. 1985. Nature 314:452-454). Alternatively,techniques described for the production of single chain antibodies canbe adapted, using methods known in the art, to produce single chainantibodies specific for the polypeptides and fragments of thisinvention. Antibodies with related specificity, but of distinctidiotypic composition, can be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton 1991. Proc. Natl. Acad.Sci. 88:11120-3).

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity for the proteins and peptides of thisinvention. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificity are well known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween an antigen and its specific antibody (e.g., antigen/antibodycomplex formation). For example, a two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the proteins or peptides of this inventioncan be used, as well as a competitive binding assay.

The present invention is more particularly described in the followingexamples, which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES I. The Semaphorin 6D Receptor on T Cells is Required forActivation of CD4⁺ T Cells

Mice. All experiments were performed with 8-12 week old C57BL/6 micefrom Jackson Labs. OT-II mice which express the OVA[323-339]-specificTCR transgene on the C57BL/6 background were generous gifts from M.Croft. All animal procedures were conducted in complete compliance withthe NIH Guide for the Care and Use of Laboratory Animals, approved bythe Institutional Animal Care and Use Committee of the University ofNorth Carolina, Chapel Hill.

Cells. Murine bone marrow derived dendritic cells (BMDCs) were isolatedfrom bone marrow and grown in vitro for maturation. Briefly cells weregrown in GM-CSF and IL-4 for 10 days before maturing with 20 ng/ml TNF-αfor 2 additional days.

Magnetic Bead isolation of TCR Tg OTII T cells. Splenic T cells wereisolated from OTII mice based on expression of CD4. Magnetic beadpurifications were performed according to the protocol provided byMiltenyi Biotec (Auburn, Calif.). Briefly, splenocytes isolated from B6or OTII mice were incubated with anti-mouse CD4 antibody conjugated withPE (BD PharMingen, San Diego, Calif.). Spleen cell samples were thenincubated with anti-PE antibody coated magnetic beads (Miltenyi Biotec,Auburn, Calif.) and cells positively selected by passage through LScolumns attached to magnetic separators. Flow through and eluentfractions were collected following Miltenyi protocol guidelines.

SYBR Green Real-Time PCR. SYBR Green qPCR Rox mix (Abgene) was used forall quantitative PCR experiments. The following cycle conditions wereused: Stage 1) 50°, for 2 minutes; Stage 2) 95°, for 15 minutes; Stage3) 95° for 15 seconds, 56-57° for 15-30 seconds, 72° for 15-30 seconds,repeat 40×; Stage 4) dissociation curve. The relative level ofexpression for each primer target was calculated via (ΔΔCT)×1000. Targetgenes were calculated in reference to β-actin for each sample. Theβ-actin primers used were: (Forward) 5′-agggctatgctctccctcac-3′ (SEQ IDNO:3) and (Reverse) 5′-ctctcagctgtggtggtgaa-3′ (SEQ ID NO:4). The Sema6Dprimers used were: (Forward) 5′-cagaagcatgggagatggat-3′ (SEQ ID NO:5)and (Reverse) 5′-gccacccatgtcgtttttac-3′ (SEQ ID NO:6).

Cloning and production of mouse Semaphorin 6D-Ig fusion protein(Sema6D-Ig). Sema6D cDNA was obtained via reverse transcriptionreaction, according to manufacturer's instruction, utilizing SuperscriptIII (Invitrogen) and an RNA sample isolated from the brain of C57BL/6Jmice. Sema6D has multiple isoforms that differ slightly in theextracellular region between the Sema domain and the trans-membranedomain. Thus, to obtain a full length cDNA of Sema6D, the forward primer(5′-atggggttccttctgctttggtt) (SEQ ID NO:7) and reverse primer(3′-ctagtacgtgtacttgttcagtggtctg) (SEQ ID NO:8) were designed utilizingcurrent mRNA sequence for Sema6D contained within the GenBank database.PCR utilizing heat-stable DNA polymerase LATaq (TAKARA) followed by 0.8%agarose gel electrophoresis, produced a band of approximately 3 kb. The3 kb DNA band was isolated and cloned into the pCR2.1 TOPO vector(Invitrogen). Multiple sequencing reactions (UNC-CH genomics corefacility) verified that the cloned DNA sequence was identical to thefull-length sequence of Sema6D isoform 6 (Sema6D-6).

Isolation of a cDNA fragment encoding the extracellular region of mouseSema6D-6 (amino acids 1-652) was obtained via PCR amplificationutilizing the full length Sema6D-6 cloned into the pCR2.1 TOPO vector.The forward primer (5′-gcggatatcgccacccatggggttccttctgctttggttct) (SEQID NO:9) was designed to include a HindIII restriction endonuclease andthe reverse primer (5′-gcgggatccagcggcaaacacgcaggtgatgagga) (SEQ ID NO:10) was designed with a BamHI restriction endonuclease site. The PCRproduct was gel purified and digested by HindIII and BamHI restrictionendonucleases (New England Biolabs). The digested fragment containingmost of the extracellular region of Sema6D (Sema6DEC) was subcloned intoa modified pcDNA3.1 vector (Invitrogen) containing a human IgG1 fragment(Hinge-CH2-CH3). For transient expression, the sequenced SEMA6DEC-Igplasmid was transfected into the COS-7 cell line (ATCC CRL-1651) via astandard calcium phosphate transfection protocol. Serum containing DMEMmedium was substituted with a serum-free DMEM medium at 48 hour posttransfection. The supernatant containing SEMA6DEC-Ig protein washarvested at 48-72 hours after transfection and purified by protein Aaffinity chromatography. Expression and secretion of SEMA6DEC-Ig wasverified by immunoprecipitation followed by western blot analysis. Fivemilliliters of the supernatant were removed from Sema6DEC-Ig-transfectedCOS-7 cells cultured in serum-free DMEM 48 hours post transfection, andincubated with protein A/G agarose beads (Promega). Subsequent westernblotting using anti-human IgG-HRP indicated a clear band ofapproximately 100 kDa and no other major bands that might representeither degradation products or contaminating proteins.

Generation of stable expression cells was performed via co-transfectionof SEMA6DEC-Ig plasmid and a mouse dihydrofolate reductase (DHFR)encoding expression vector pSV2-dhfr (ATCC 37146) into DHFR-Chinesehamster ovary cells (CHO/DG44, Invitrogen) at a 20:1 ratio(weight:weight) through electroporation technique (300V, 960 uF,Bio-rad). Stable Sema6D-Ig expressing CHO cell clones were selected inExcell 302 serum free CHO medium (JRH Biosciences) supplemented withL-glutamine (Invitrogen) and 100 nM methotrexate (MTX, Sigma). Sema6D-Igproduced by the CHO cells was harvested from large-scale cultures viaprotein A affinity chromatography followed by gel filtrationchromatography purification (Biosilect 400, Bio-rad).

Semi-quantitative RT-PCR of Type III semaphorin transcripts. Total RNAwas isolated from DO11.10 T cells with or without activation, 3B11 cellsat day 0, day 3, day 5 and day 7 of maturation, and brain cells usingTRIzol Reagent (Invitrogen). One microgram of RNA of each sample wasreverse transcribed using MMLV reverse transcriptase (Invitrogen).Semaphorin 3A, 3B, 3C, 3D and 3E transcripts were assessed using Taqpolymerase (Invitrogen) and semi-quantitative PCR (22 cycles).Standardization of cDNA amounts was analyzed via PCR for 18S RNA.Sequences of the primers for the class III semaphorins are as follows:

Semaphorin 3A, (forward) (SEQ ID NO: 11) 5′-CGGGACTTCGCTATCTTCAG-3′ and(reverse) (SEQ ID NO: 12) 5′-AGCATGAGTGGCTTTTCCAG-3′; Semaphorin 3B,(forward) (SEQ ID NO: 13) 5′-GCTGTCTTCTCCACCTCCAG-3′ and (reverse) (SEQID NO: 14) 5′-GGTTCCGACCAAACTGGATA-3′; Semaphorin 3C, (forward) (SEQ IDNO: 15) 5′-TCGGCAGTGTGTGTGTATCA-3′ and (reverse) (SEQ ID NO: 16)5′-CCTTCTGTGGATGGGGTAGA-3′; Semaphorin 3D, (forward) (SEQ ID NO: 17)5′-ATGGCTGATATCCGAGCAGT-3′ and (reverse) (SEQ ID NO: 18)5′-TTCTCTTGAAGGTCGGTGCT-3′; and Semaphorin 3E, (forward) (SEQ ID NO: 19)5′-GAGGCCATGCTGTATGTGTG-3′ and (reverse) (SEQ ID NO: 20)5′-CGTCATCGGGTAATCTTTGG-3′.

Flow Cytometry. Following splenic or BM isolation, cells were suspendedin ammonium chloride-Tris buffer (ACT) for 3 minutes at 37° C. to removeRBC. ACT treatment was performed with carboxyfluorescein diacetatesuccinimidyl ester (CFDAse, Molecular Probes, Eugene, Oreg.) labeledcells. Following ACT treatment, cells were washed and resuspended in 5%BCS in BSS and stained with the appropriate antibodies as described. Forall studies, non-specific staining was reduced by addition of FcRblocking antibody and unlabeled Rat/Hamster Ig. Incubation withbiotinylated antibodies was followed by incubation with Streptavidin-PE,PerCP or APC (BD PharMingen, San Diego, Calif.). Primary antibodyincubations were for a minimum of 30 minutes at 4° C. followed bywashing in BCS/BSS. Secondary antibody incubations were for a maximum of15 minutes at 4° C. followed by washing in BCS/BSS. Stained cells wereeither analyzed immediately or fixed with 1% formaldehyde in 1.25×PBS.Staining was quantified with a Becton Dickinson FACSCalibur. A minimumof 50,000 events was collected and fluorescence signals detected viafour-decade logarithmic amplification except for FSC and SSC which weredetected via a linear scale. Spectral overlap compensation was made withsingle-color stained samples for each detection channel. For eachexperiment, data were analyzed using FlowJo software (Treestar, Calif.).

In vitro CD3/CD28 stimulation. For stimulation of T cells, 5 μg/ml ofanti-mouse CD3 and anti-mouse CD28 were added in PBS to cell cultureplates for overnight coating at 4° C. For a 6 well plate, 1 ml/well wasused. Following the overnight incubation, the plates were washed 3× withPBS or complete medium (cRPMI: RPMI+serum). Primary T cells isolatedfrom spleens were incubated at 1×10⁶ cells/ml in 2 mls per well of a 6well coated plate.

Ovalbumin (OVA) (whole protein or peptide) loading of DCs. BMDCs,cultured for up to 10 days in cRPMI supplemented with GMCSF and IL4,were resuspended at a concentration of 1×10⁶ cells in 1 ml of cRPMI with10 μg/mL whole OVA protein or peptide. The cells were incubated for 12hrs at 37° C. with rotation. Following the incubation, the cells werewashed 2× in cRPMI.

Adoptive transfer. Following isolation of splenocytes or BMDCs frommice, RBCs were lysed via incubation with ACT. The percentage of Tg OTIIT cells within a population was determined by staining 2×10⁵ cells withanti-Vα2 and anti-Vβ5 in 5% BCS in BSS at 4° C. and analyzed via aBecton Dickinson FACSCalibur cell sorter. For each primary transfer,3×10⁶ T cells and BMDCs were injected via tail vein into B6 recipientmice. Typically, three mice were used per experimental group.

CFSE labeling of T cells. T cells labeled with carboxyfluoresceindiacetate succinimidyl ester (CFDAse or CFSE; Molecular Probes, Eugene,Oreg.) were incubated at 37° C. for 10 minutes in serum free RPMI. Thefinal concentration of CFSE used was 15 μM in RPMI with 10-20 millioncells per ml. Following incubation with CFSE, the T cells were washed incRPMI. Experimental conditions permitting, cells utilized for CFSElabeling were not treated with ASC red blood cell lysis buffer at thetime of isolation.

Activation of T cells by co-culture with Ag-loaded BMDCs. For in vitroactivation, OTII TCR Tg (OVA-specific) T cells were incubated withimmature BMDCs at a ratio of 1:1 in RPMI. OTII T cells were isolatedfrom the spleens of Tg mice and purified by negative selection with Tenrichment columns (R&D systems). Isolated T cells were labeled withCFSE or unlabeled prior to culture. BMDCs were either unloaded or loadedwith OVA antigen (Ag) prior to culture. Approximately 0.5×10⁶ T cellsand BMDCs were cultured in 1 ml per well of a 24 well plate. At theculture initiation, IL4 & GM-CSF were added at a concentration of 5ηg/mL.

Use of anti-Sema6D antibody or the Sema6D-Ig fusion protein to block thefunctional activation of T cells. Antibodies for blocking interactionsbetween the T cells and BMDCs, such as anti-Sema6D Ab, were used at afinal concentration of 10 μg/ml. The Sema6D-Ig fusion protein was usedat a final concentration of 5 μg/ml. One day following initiation, cellcultures were supplemented with 1 ml of cRPMI. The cultured cells wereanalyzed by flow cytometry for indications of T cell activation viaproliferation and expression of activation markers as described herein.

Activated CD4⁺ T cells express Semaphorin 6D in vitro. CD4⁺ T cells wereisolated from splenocytes by magnetic bead separation and activated invitro by anti-CD3 and anti-CD28 stimulation. Splenic CD4⁺ T cells wereisolated by magnetic bead selection to a purity of greater than 90%.Purified T cells were cultured with plate bound anti-CD3 and -CD28antibodies for stimulation. RNA was isolated from cultures at 12, 24 and48 hr post initiation and analyzed by qPCR for Semaphorin 6D (Sema6D)expression. Following 12 hrs of stimulation, expression of Sema6D mRNAwas increased as measured by qPCR, and this enhancement continued untilat least 48 hrs post activation. Protein expression of Sema6D onactivated CD4⁺ T cells was also examined by flow cytometry. Following 96hrs of anti-CD3 and anti-CD28 stimulation, enhanced expression of CD25and CD44 was detected on CD3⁺CD4⁺ T cells, indicative of theiractivation. Concurrently, upregulation of Sema6D was observed onCD3⁺CD4⁺ T cells following 96 hrs of stimulation. Isotype-matchedcontrol Ig showed no such increase. Thus, activation of CD4⁺ T cells viastimulation of CD3 and CD28 results in an upregulated expression ofSema6D at the cell surface. In contrast, measurements of Semaphorin 3Ato 3F by the highly sensitive RT-PCR failed to detect any signals inresting or activated T cells. Semi-quantitative RT-PCR analysis revealedthat OTII T cells did not express detectable levels of any type 3semaphorins, including Sema 3A-E. Semaphorin 3 expression was detectedin brain samples, used as positive controls.

DC mediated activation of Tg OTII T cells results in Sema6D expressionin vivo. Although expression of Sema6D in vitro via anti-CD3 and —CD28stimulation was observed, it remained uncertain whether this resultreflected the physiological reality of in vivo T cell activation. Toexamine the in vivo situation, the TCR transgenic (Tg) mouse line, OTII,whose CD4 T cells express a TCR specific for the OVA antigen, was used.OTII Tg T cells were isolated from splenocytes and adoptivelytransferred to recipient mice with either OVA-loaded DCs (immune) orun-loaded DCs (naïve). The recipient mouse splenocytes were harvested atdays 2, 3 and 4 post adoptive transfer and the cells were analyzed byflow cytometry. The activation and expansion of the OTII T cells werevisualized as an expansion of the population of T cells expressing theTg TCR Vα2 and Vβ5 chains. Proliferation of the OTII T cells wasobserved in vivo by day 2 and peaked at day 4, representing anapproximately 5-fold expansion in immune vs. naïve mice. Concurrently,on day 4, the expression of CD25 was upregulated on OTII T cells fromimmune mice vs. naïve mice. This was accompanied by an upregulation ofSema6D on activated OTII T cells in vivo vs. naïve mice. Thus, in aphysiologically relevant system of in vivo antigen presenting cellmediated T stimulation, enhanced expression of Sema6D on activated CD4⁺T cells was observed, confirming the induction of Sema6D during T cellactivation.

Blocking Sema6D antibody inhibits DC mediated OTII T cell proliferationand activation. To examine the functional consequence of Sema6Dexpression on T cells, an in vitro DC-mediated T cell activation assaywas used. Tg OTII T cells were cultured with OVA loaded BMDCs (OVA-BMDC)or unloaded BMDCs (BMDC). Following isolation but prior to co-culture,the OTII T cells were labeled with CFSE to enable monitoring ofactivation-induced proliferation. At days 2, 6 and 7 post cultureinitiation, cultured cells were collected and analyzed by flowcytometry. Activation is associated with proliferation, which results ina serial dilution of CFSE staining intensity with each cell division.Thus a pattern of serially diluted CFSE staining is indicative of T cellactivation. OTII T cells cultured control exhibited little change, whileOTII T cells incubated with OVA-BMDC cells displayed activation-inducedproliferation as measured by a dilution of CFSE intensity. Initially, asmall amount of proliferation was observed on day 2 post-culture of Vβ5⁺(OTII) T cells with OVA-BMDC but not control BMDC (0.94% Vβ5⁺CFSE^(low)vs. 0.064%). By day 7 of co-culture, the OTII T cells incubated withOVA-BMDC proliferated greatly compared with those cultured with controlBMDC, representing a greater than 11 fold induction. The proliferatingcells observed on day 7 were TCR⁺CD4⁺CD8⁻ T cells, indicative of theOTII Tg T cell phenotype.

Significantly, when OTII T cells were cultured with OVA-BMDC in thepresence of an antibody to block Sema6D (Sema6D Ab), the proliferationof the T cells was abrogated. While proliferation on day 6 and 7 wasmarkedly reduced, the initial level of proliferation observed on day 2was comparable to cultures with a control antibody (Ctrl Ab). Thus,while early survival may be unaffected, optimal proliferation andhomeostasis of the Vβ5⁺ OTII T cells were inhibited by Sema6D blockadeat both days 6 and 7.

The expression of Sema6D on in vitro activated OTII T cells was alsoexamined and expression on both unactivated and activated T cells by day7 was observed. BMDCs that were loaded (OVA-BMDC) or unloaded (BMDC)with whole OVA protein were cultured with purified OTII T cells invitro. Prior to culture initiation, OTII T cells were labeled with CFSE.Antigen positive cultures were also treated with either a Sema6Dblocking antibody or a control antibody. As expected, addition ofblocking antibody significantly inhibited the detection of Sema6Dexpression compared with control antibody or unactivated cultures.

Finally, the ability of blocking Sema6D Ab to inhibit the appearance ofan activated T cell phenotype was examined. Expression levels of CD25,CD62L, CD69, CD154 and CD44 were analyzed. For all the phenotypicmarkers analyzed, blocking Sema6D antibody inhibited the accumulation orappearance of activated OTII Tg T cells while isotype-control antibodydid not. The low number of cells displaying an activated phenotype inthe Sema6D Ab treated group may reflect an initial activation andproliferation that occurs in the presence of Sema6D Ab. Moreover, theredoes not appear to be reduced viability of CFSE^(bright) T cells lackingactivation makers, suggesting that Sema6D Ab affects only stimulated Tcells. These data indicate that Sema6D regulates DC mediated T cellproliferation, activation and survival.

Sema6D-Ig inhibits BMDC mediated T cell activation. To furthercharacterize the function of Sema6D, a hybrid of a cDNA fragmentencoding the extracellular region of mouse Sema6D-6 (amino acids 1-652)and a human IgG1 fragment (hinge-CH2-CH3) was produced, resulting in aSema6D-Ig fusion protein. This fusion protein was used along with theanti-Sema6D antibody in an experimental procedure as described herein.BMDCs that were loaded (OVA-BMDC) or unloaded (BMDC) with whole OVAprotein were cultured with purified OTII T cells in vitro. Prior toculture initiation, OTII T cells were labeled with CFSE. Antigenpositive cultures were also treated with a control antibody, a MHC classII blocking antibody, a Sema6D blocking antibody or the Sema6D-Ig fusionprotein. At day 5 post culture initiation, the proliferation of CD4⁺ Tcells was analyzed. Utilizing Sema6D-Ig as a blocking reagentadministered to in vitro cultures, inhibition of BMDC mediated T cellproliferation was observed, as compared to a control antibody treatedgroup (5.15% CFSE^(low)CD4⁺ vs. 29%). The level of inhibition with theSema6D-Ig fusion protein was comparable to inhibition via theanti-Sema6D Ab. As a control, treatment with a blocking antibody for MHCII resulted in complete inhibition of T cell activation.

These studies demonstrate that activated T cells express high levels ofSemaphorin 6D both in vitro and in vivo and that inhibition of Sema6D,via treatment with a blocking Ab or Sema6D-Ig, significantly inhibitsdendritic cell mediated T cell activation. These data demonstrate thatSema6D represents an important novel receptor for the regulation of Tcell immunity.

These studies further indicate that Sema6D inhibitors may reduce thesurvival of activated T cells only and do not appear to function asgeneral inhibitors of T cell survival. In Sema6D Ab treated cultures(OVA-BMDC+Sema6D), the viability of non-dividing CFSE^(bright) and TCR⁺or CD4⁺ cells did not appear to be affected when compared with T cellsfrom naïve (BMDC) cultures (72.5 vs. 72.4%; and 66.1 vs. 67.2%respectively). Thus, in the case of autoimmunity, blocking Sema6D wouldallow for the specific targeting of activated autoimmune T cells whileallowing unactivated, non-autoimmune T cells to persist. A similarmethod could be applied to transplant patients to induce a tolerizingeffect on rejecting T cells. Alternatively, stimulating activated Tcells via agonist ligand binding of Sema6D, could lead to enhancedvaccine efficacy or even enhanced tumor rejection via stimulation ofanti-tumor T cells.

II. Blocking Sema6D with the Sema6D-Ig Fusion Protein Caused a DelayedInhibition of T Cell Activation

This was tested by assaying the phosphorylation of three T cellactivating molecules, CrkL, LAT and CD3ζ. CD3ζ phosphorylation is anearly event in T cell activation that occurs proximal to the T cellreceptor. Its activation as indicated by phosphorylation is not alteredby Sema6D-Ig inhibition. In the studies conducted, Sema6D was shown toregulate endogenous T cell signaling during late-stage activation. OTIIT cells were co-cultured with DCs loaded with OVA antigen (OVA-DC) orunloaded DCs (DC). Antigen positive cultures were treated with Sema6D-Ig(S6D-Ig) or human IgG1 (hIgG1). OTII T cells co-cultured with DCs wereanalyzed by phosphor-specific flow cytometry for endogenous signalingpathways. FACS analysis of phosphorylated CrkL in TCR⁺ OTII T cells wascarried out at days 3 and 6 of co-culture with DCs. FACS analysis ofphosphorylated LAT in TCR⁺ OTII T cells was also carried out at days 3and 6. FACS analysis of phosphorylated CD3ζ in TCR⁺ OTII T cells wasalso carried out at days 3 and 6 of co-culture.

Phosphorylation of CrkL is an indication of c-Abl activation, and it wasinhibited by Sema6D-Ig inhibition but only at a late time point (day 6but not day 3). LAT phosphorylation which lies downstream of c-Ablphosphorylation was inhibited by Sema6D-Ig at a late time point (day 6but not day 3). These results indicate that Sema6D signaling is mostrelevant late during T cell activation. Further it lies upstream ofc-Abl, CrkL and LAT phosphorylation but does not affect CD3ζphosphorylation. Thus blocking Sema6D-Ig is a mechanism to block late Tcell activation, which provides a different intervening point from mostimmune clinical biologics used in the market.

III. Expression of Sema6D on B Cells

This is of particular interest as recently anti-B cell antibodies havebeen effective in both reducing autoimmunity in people, and reducing Blymphoma growth. Sema6D was found to be expressed by B cells to asimilar extent as T cells, in both mouse and human (FIGS. 1 a,b).Furthermore, expression was shown in four different types of leukemia(FIG. 1 c). Blocking Sema6D was shown to reduce T cell proliferation andB cell proliferation. This indicates that blocking Sema6D can reducelymphocyte survival, which is important in the control of autoimmunity,but also in the control of transformed B and T lymphomas/leukemia.

Expression of Sema6D protein in B cells is enhanced during an immuneresponse. To explore if the expression of Sema6D is enhanced during Bcell activation (a state similar to auto-activated B cells implicated inautoimmune diseases such as systemic lupus and arthritis, andtransformed B cells found in leukemia and lymphomas), B cells activatedin vivo were tested with antigens and antigen presenting cells. DCsmatured for 8 days in vitro were loaded with whole OVA protein and thentransferred by i.v. injection with OTII TCR transgenic (OVA specific) Tcells to recipient B6 mice. At day 4 post-transfer, recipient mousesplenocytes were isolated and analyzed by flow cytometry for theexpression of Sema6D. Splenocytes were incubated with anti-CD45, -B220and -Sema6D antibodies. B220⁺ CD45⁺ splenocytes were gated and analyzedfor expression of Sema6D. The percentage of B220⁺ Sema6D⁺ cells wasdisplayed for splenocytes from naïve and immune animals. These studiesshowed that in vivo activated B cells expressed significantly higherlevels of Sema6D. In these experiments, B cells were marked with B220,and they showed elevated Sema6D after antigen stimulation. This suggeststhat Sema6D-Ig molecules might selectively target activated B cells andtransformed B cells, but not naïve resting B cells.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents, patent publications andnon-patent publications are referenced. The disclosures of these patentsand publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

REFERENCES

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TABLE 1 Nucleotide and amino acid sequences of the Sema6D protein of theinvention and a fusion protein of the invention. SEQ ID Accession No.Organism Definition NO: NM_020858 Homo sapiens Homo sapiens sema domain,transmembrane 21 domain (TM), and cytoplasmic domain, (semaphorin) 6D(SEMA6D), transcript variant 1, mRNA. NP_065909 Homo sapiens semaphorin6D isoform 1 precursor 22 NM_153616 Homo sapiens Homo sapiens semadomain, transmembrane 23 domain (TM), and cytoplasmic domain,(semaphorin) 6D (SEMA6D), transcript variant 2, mRNA NP_705869 Homosapiens semaphorin 6D isoform 2 precursor 24 NM_153617 Homo sapiens Homosapiens sema domain, transmembrane 25 domain (TM), and cytoplasmicdomain, (semaphorin) 6D (SEMA6D), transcript variant 3, mRNA NP_705870Homo sapiens semaphorin 6D isoform 3 precursor 26 NM_153618 Homo sapiensHomo sapiens sema domain, transmembrane 27 domain (TM), and cytoplasmicdomain, (semaphorin) 6D (SEMA6D), transcript variant 4, mRNA NP_705871Homo sapiens semaphorin 6D isoform 4 precursor 28 NM_153619 Homo sapiensHomo sapiens sema domain, transmembrane 29 domain (TM), and cytoplasmicdomain, (semaphorin) 6D (SEMA6D), transcript variant 5, mRNA NP_705872Homo sapiens semaphorin 6D isoform 5 precursor 30 NM_024966 Homo sapiensHomo sapiens sema domain, transmembrane 31 domain (TM), and cytoplasmicdomain, (semaphorin) 6D (SEMA6D), transcript variant 6, mRNA NP_079242Homo sapiens semaphorin 6D isoform 6 precursor 32 NM_172537 Mus musculusMus musculus sema domain, transmembrane 33 domain (TM), and cytoplasmicdomain, (semaphorin) 6D (Sema6d), transcript variant 1, mRNA n/a Musmusculus CDS of NM_172537 34 NP_766125 Mus musculus sema domain,transmembrane domain (TM), 35 and cytoplasmic domain, (semaphorin) 6Disoform 1 n/a Mus musculus CDS of NM_199238 36 NP_954708 Mus musculussema domain, transmembrane domain (TM), 37 and cytoplasmic domain,(semaphorin) 6D isoform 2 n/a Mus musculus CDS of NM_199241 38 NP_954711Mus musculus sema domain, transmembrane domain (TM), 39 and cytoplasmicdomain, (semaphorin) 6D isoform 4 n/a Mus musculus CDS of NM_199239 47NP_954709 Mus musculus sema domain, transmembrane domain (TM), 48 andcytoplasmic domain, (semaphorin) 6D isoform 5 n/a Mus musculus CDS ofNM_199240 49 NP_954710 Mus musculus sema domain, transmembrane domain(TM), 50 and cytoplasmic domain, (semaphorin) 6D isoform 6 n/a Musmusculus CDS of Sema6D-6 51 BC098887 Danio rerio Danio rerio semadomain, transmembrane 52 domain (TM), and cytoplasmic domain,(semaphorin) 6D, mRNA AAH98887 Danio rerio Sema domain, transmembranedomain (TM), 53 and cytoplasmic domain, (semaphorin) 6D XM_230583 RattusPREDICTED: Rattus norvegicus sema 54 norvegius domain, transmembranedomain (TM), and cytoplasmic domain, (semaphorin) 6D (predicted)(Sema6d_predicted), mRNA XP_230583 Rattus PREDICTED: similar to semadomain, 55 norvegius transmembrane domain (TM), and cytoplasmic domain,(semaphorin) 6D isoform 4 XM_596649 Bos taurus PREDICTED: Bos taurussimilar to 56 semaphorin 6D, transcript variant 5 (LOC518458), mRNAXP_596649 Bos taurus PREDICTED: similar to semaphorin 6D 57 isoform 5n/a Artificial CDS of Murine sema6D-Ig fusion protein 58

TABLE 2 PCR and Sequencing primers Primer sequence SEQ ID NO:ATGGGGTTCCTTCTGCTTTGGTT (offset: 1; 7 23 nt)CTAGTACGTGTACTTGTTCAGTGGTCTG (offset: 8 2997; 28 nt)AAAGCAGAAGGAACCCCATGGTT (Rev. -838) 40 ACCAGGTAGCTAAGTGGGACTTCTG (For.761-) 41 TGACACCCTGGCTTTCATCAAGT (For. 1161-) 42AAAGTCTTGCATTGCATCACGTGAC (For. 1566-) 43 CCAATCAGATGGTCCACATGAA (For.1964-) 44 ATGAAGAGCCACTCTGAGAAGGC (For. 2362-) 45 TAACCGGGAGGCATCTCTATAC(For. 2769-) 46

1. A method of reducing T cell activation in a subject, comprisingadministering to a subject in need of reduced T cell activation aneffective amount of an inhibitor of Semaphorin 6D (Sema6D) activity on Tcells.
 2. The method of claim 1, wherein the subject is atransplantation patient, a subject having an autoimmune disease or atrisk of having an autoimmune-disease (e.g., SLE, MS, RA, type Idiabetes), and/or a subject having an inflammatory response or at riskof having an inflammatory response.
 3. The method of claim 1, whereinthe inhibitor of Sema6D activity is an antibody that specifically bindsSema6D.
 4. The method of claim 1, wherein the inhibitor of Sema6Dactivity is administered in combination with another anti-T celltherapeutic, either simultaneously, before and/or after administrationof the inhibitor of Sema6D activity.
 5. A method of increasing T cellactivation in a subject, comprising administering to a subject in needof increased T cell activation an effective amount of an enhancer ofSemaphorin 6D (Sema6D) activity on T cells.
 6. The method of claim 5,wherein the enhancer of Sema6D activity is administered in combinationwith another T cell activating therapeutic, either simultaneously,before and/or after administration of the enhancer of Sema6D activity.7. A method of identifying an activated T cell, comprising detectingSema6D on the surface of the T cell.
 8. A method of identifying anactivated T cell, comprising detecting messenger RNA encoding Sema6D inthe T cell.
 9. A method of monitoring T cell activation over time,comprising detecting Sema6D on the surface of a T cell over time andmeasuring changes in the amount of Sema6D on the surface of a T cellover time.
 10. A method of monitoring T cell activation over time,comprising detecting messenger RNA encoding. Sema6D in a T cell overtime and measuring changes in the amount of messenger RNA encodingSema6D in the T cell over time.
 11. A method of identifying a substancehaving an inhibitory effect on Sema6D activity and/or having aninhibitory effect on T cell activation, comprising contacting thesubstance with T cells under conditions whereby Sema6D activity and/or Tcell activation can occur and measuring the amount of Sema6D activityand/or T cell activation in the presence and in the absence of thesubstance; whereby a decrease in Sema6D activity and/or T cellactivation in the presence of the substance as compared to the amount ofSema6D activity and or/T cell activation in the absence of the substanceidentifies a substance having the ability to inhibit Sema6D activityand/or T cell activation.
 12. A method of identifying a substance havingan enhancing effect on Sema6D activity and/or T cell activation,comprising contacting the substance with T cells under conditionswhereby Sema6D activity and/or T cell activation can occur and measuringthe amount of Sema6D activity and/or T cell activation in the presenceand in the absence of the substance; whereby an increase in Sema6Dactivity and/or T cell activation in the presence of the substance ascompared to the amount of Sema6D activity and/or T cell activation inthe absence of the substance identifies a substance having the abilityto enhance Sema6D activity and/or T cell activation.
 13. A method ofreducing B cell activation in a subject, comprising administering to asubject in need of reduced B cell activation an effective amount of aninhibitor of Semaphorin 6D (Sema6D) activity on B cells.
 14. The methodof claim 13, wherein the subject is a transplantation patient, a subjecthaving an autoimmune disease or at risk of having an autoimmune disease(e.g., SLE, MS, RA, type I diabetes), and/or a subject having aninflammatory response or at risk of having an inflammatory response. 15.The method of claim 13, wherein the inhibitor of Sema6D activity is anantibody that specifically binds Sema6D.
 16. The method of claim 13,wherein the inhibitor of Sema6D activity is administered in combinationwith another anti-B cell therapeutic, either simultaneously, beforeand/or after administration of the inhibitor of Sema6D activity.
 17. Amethod of increasing B cell activation in a subject, comprisingadministering to a subject in need of increased B cell activation aneffective amount of an enhancer of Semaphorin 6D (Sema6D) activity on Bcells.
 18. The method of claim 17, wherein the enhancer of Sema6Dactivity is administered in combination with another B cell activatingtherapeutic, either simultaneously, before and/or after administrationof the enhancer of Sema6D activity.
 19. A method of identifying anactivated B cell, comprising detecting Sema6D on the surface of the Bcell.
 20. A method of identifying an activated B cell, comprisingdetecting messenger RNA encoding Sema6D in the B cell.
 21. A method ofmonitoring B cell activation over time, comprising detecting Sema6D onthe surface of a B cell over time and measuring changes in the amount ofSema6D on the surface of a B cell over time.
 22. A method of monitoringB cell activation over time, comprising detecting messenger RNA encodingSema6D in a B cell over time and measuring changes in the amount ofmessenger RNA encoding Sema6D in the B cell over time.
 23. A method ofidentifying a substance having an inhibitory effect on Sema6D activityand/or having an inhibitory effect on B cell activation, comprisingcontacting the substance with B cells under conditions whereby Sema6Dactivity and/or B cell activation can occur and measuring the amount ofSema6D activity and/or B cell activation in the presence and in theabsence of the substance; whereby a decrease in Sema6D activity and/or Bcell activation in the presence of the substance as compared to theamount of Sema6D activity and or/B cell activation in the absence of thesubstance identifies a substance having the ability to inhibit Sema6Dactivity and/or B cell activation.
 24. A method of identifying asubstance having an enhancing effect on Sema6D activity and/or B cellactivation, comprising contacting the substance with B cells underconditions whereby Sema6D activity and/or B cell activation can occurand measuring the amount of Sema6D activity and/or B cell activation inthe presence and in the absence of the substance; whereby an increase inSema6D activity and/or B cell activation in the presence of thesubstance as compared to the amount of Sema6D activity and/or B cellactivation in the absence of the substance identifies a substance havingthe ability to enhance Sema6D activity and/or B cell activation.
 25. Amethod of treating a B cell-related disorder in a subject, comprisingadministering to the subject a therapeutic amount of an inhibitor ofSemaphorin 6D (Sema6D) activity on B cells.
 26. The method of claim 25,wherein the disorder is selected from the group consisting of leukemia,lymphoma, an autoimmune disorder, inflammatory response, transplantationrejection and any combination thereof.
 27. A method of treating a Tcell-related disorder in a subject, comprising administering to thesubject a therapeutic amount of an inhibitor of Semaphorin 6D (Sema6D)activity on B cells.
 28. The method of claim 27, wherein the disorder isselected from the group consisting of leukemia, lymphoma, autoimmunedisease, inflammatory response, transplantation rejection and anycombination thereof.
 29. A method of treating a white blood cell-relateddisorder in a subject, comprising administering to the subject atherapeutic amount of an inhibitor of Semaphorin 6D (Sema6D) activity onwhite blood cells.
 30. The method of claim 29, wherein the disorder isselected from the group consisting of leukemia (e.g., chronicmyelogenous leukemia; promyelocytic leukemia), lymphoma, an autoimmunedisorder and any combination thereof.